WO2023017251A1 - Primary cell extraction and preservation from fluids - Google Patents

Abstract

Provided are methods for isolating viable mammalian cells from a biofluid sample, suitably from urine. Such a method includes providing a device comprising a receptacle for receiving the fluid sample, and a filtration mechanism comprising one or more filters, the filter comprising polycarbonate, polyester, polyether sulfone, stabilised/ regenerated cellulose and polyethylene terephthalate; placing a fluid sample in the receptacle; passing the fluid sample through the filter by the action of gravity and/or by the application of positive pressure to the fluid sample; and contacting the filter with a culture medium suitable for supporting cells. Associated devices are also provided.

Description

PRIMARY CELL EXTRACTION AND PRESERVATION FROM FLUIDS

FIELD OF INVENTION

The invention relates to apparatuses and methods for separating cells from liquids and their preservation.

BACKGROUND

Much of academic and industrial biological research and clinical diagnosis relies on cell culture experiments across various disciplines. Human or mammalian body fluids, aka. bodily fluids or biological fluids (e.g. urine, breastmilk, semen, etc.) have recently emerged as a new non-invasive source of patient-specific cells that can be expanded and studied in the laboratory for a variety of purposes, including personalised disease modelling and precision medicine application. Urine of the healthy adults contains between 2.5-7.5 live cells per 100ml, and these are capable of extensive proliferation in vitro yielding millions of cells within 2-4 weeks (Zhang et al, 2008, Wu et al, 2011). The reported success rate in initiating and expanding cells from urine for healthy donors ranges from 10% (n=38, adults) to 19% (n=48, 4-13 years old) to 52% (n=24, adults) and 73% (n=57, adults), depending on the publication (Wilmer et al 2010, Wilmer et al 2005, Dorrenhaus, 2000, Zhou et al, 2012). Methodological differences in how urine-derived cells are extracted and cultured may explain some of the variation reported.

Standard methods of processing urine samples require centrifugation in laboratory settings shortly after sample collection It is thought that cells deteriorate rapidly when suspended in urine because they are easily harmed by toxins, need nutrients to survive and prefer a pH around 7. Urine contains metabolic waste products, lacks many nutrients and has a varying range of pH (5.0-8.0), as well as proteases that may compromise cell integrity. Rapid processing of fresh samples is generally accepted as a pre-requisite for successful cell derivation, with the current cut-off of 4hrs.

Currently accepted protocols for urine processing requires a rapid 2-step centrifugation, which in turn needs transportation of batches of samples between the collection site (e.g. clinic) and a processing facility (e.g. laboratory). As a result, cells’ exposure to urine can range from 0.5 to 4hrs in published studies, which has unknown consequences on urine- derived cell viability and yields. The short shelf life of samples limits patient inclusion in studies, particularly in the fields of rare genetic diseases and paediatrics. Establishing a new, more reliable and more effective method of cell extraction from biofluids that does not rely on ready access to lab- based equipment would enable remote sourcing of cells from the donors of interest, at higher viability.

While centrifugation is an extremely common procedure used routinely to concentrate cells, the volumes are usually small (1-10ml) and the cell numbers are high (for example, 10⁶ or greater). Urine and some other biofluids are different For viable cell recovery, larger volumes need to be processed (50-100ml), and the samples have variable numbers of particles, including intact cells, cell debris, protein, mucous, and salts. As a result, inconsistent results are achieved. Despite centrifugation being used to extract cells from urine, no investigation into its efficiency to concentrate cells or potential cell loss has been conducted. There is also a range in the number of clones obtained from each sample with inter- and intra-donor variability, which is currently attributed to chance. The number of clones obtained is crucial, as it determines the number of experiments that can be done with the cells. Researchers therefore aim to process samples as soon as possible (i.e. within 15-30 minutes of collection) to minimise harmful effects of extended exposure of cells to the toxic urine environment.

Some attempts have been made to stabilise cells in urine, primarily by changing the environment chemically. Lang et al, 2013 reports a urine cell preservation method, by adding 5% Fetal Bovine Serum to the samples, reportedly extending the shelf life of samples to 12 hours at 4°C. Schutgens et al 2019 describes a method where Rho-kinase inhibitor Y-27632 and primocine (antibiotic and antifungal) are added to urine samples to preserve cells before they are centrifuged.

Urine filtration devices and associated methods in development typically lead to death of the cells during the filtration process, as the object is to analyse cell contents such as DNA/RNA for gene expression profiling, rather than to isolate viable cells for expansion.

US2016223442A1 describes biological fluid filtration assemblies for the isolation of bladder tumour cells to extract DNA/RNA. This application does not require viable cells, hence questions of damage to the cells during filtration are not addressed. Other cell filtration devices, such as those aiming to recover certain cell types from blood, are lab-based solutions, requiring some degree of training and specialised equipment, in particular given the steps required to take a blood sample. These are therefore not suitable for direct use by patients or those with lower access to laboratory resources.

Improved methods are therefore needed to extract cells of interest from liquid biopsies, with greater success and reliability at isolating viable cells, which do not require immediate processing of the samples by specialised laboratory equipment (e.g. centrifuge and sterile environment of the cell culture facilities), and which can be done by donors at their homes or other convenient location.

SUMMARY OF THE INVENTION

Devices and methods useful for isolating viable cells from biofluids, are described. These use filters and means for passing the biofluids through said filters, in order to isolate the cells. The devices and methods demonstrate improved success at isolation of viable cells compared to alternatives involving centrifugation.

In a first aspect, there is provided a method for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types. The method comprises providing a device comprising a receptacle for receiving the fluid sample, and a filtration mechanism comprising one or more filters. The filter comprises polycarbonate, polyester, polyether sulfone, stabilised/ regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate. The method further comprises placing the fluid sample in the receptacle and passing the fluid sample through the filter by the action of gravity and/or by the application of a positive pressure to the fluid sample. The filter is contacted with a culture medium suitable for supporting cells.

The biofluid sample may be or may comprise urine. The biofluid may be passed through the filter within 30 minutes of being obtained from a subject, and may be maintained at between 15 and 25°C between its production and being passed through the filter.

In some embodiments, the viable mammalian cells comprise urine-derived renal epithelial cells (URECs), and may comprise immune system cells and/or cells originating from the renal and/or urological tracts. The filter may have an average pore diameter of at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, suitably at least 5 μm. The filter may have an average pore diameter of at most 10 μm, at most 8 μm, at most 7 μm, at most 6.5 μm, at most 6 μm, at most 5.5 μm, typically at most 5 μm.

In some embodiments, the fluid sample is passed through the filter by the action of gravity alone. In some embodiments, the receptacle is compressed to apply positive pressure to the fluid sample. An air pump may be used to apply positive pressure to the fluid sample; the air pump comprising in some embodiments a compressible bladder.

The filter may comprise a polyether sulfone membrane, which may have an average pore diameter of less than about 6 μm.

In some embodiments, the fluid sample is passed through the filter with a flow rate of at least 100, at least 120, at least 140, at least 160, suitably at least 180 mL/min/cm² at 69 kPa. The fluid sample may be passed through the filter with a flow rate of at most 300, at most 280, at most 260, at most 240, at most 220, at most 210, at most 200, suitably at most 190 mL/min/cm² at 69 kPa.

In some embodiments, after the fluid sample is passed through the filter, the device is sealed to create a liquid-tight chamber. In some embodiments, after the fluid sample is passed through the filter, the filter is removed and placed within a sealable chamber which comprises a culture medium and/or which is configured to receive a culture medium.

In a second aspect, there is provided a device for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types. The device comprises a receptacle for receiving the biofluid sample and a filtration mechanism comprising at least one filter, wherein the filter comprises polyether sulfone. The filter has an average pore diameter of less than about 6 μm.

In some embodiments, the filter has an average pore diameter of greater than about 3 μm.

The receptacle may be collapsible, and may be configured such that, on collapsing, positive pressure is applied to the biofluid sample to drive it through the filter. The device may further comprise an air pump, wherein the air pump is configured to attach to the receptacle and apply a positive pressure to the biofluid sample. In some embodiments, the air pump comprises a compressible bladder.

In a third aspect, there is provided a device for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types. The device comprises a receptacle for receiving the biofluid sample, and a filtration mechanism comprising at least one filter, wherein the filter comprises polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate. The receptacle is collapsible such that on compression, positive pressure is applied to the biofluid sample to drive it through the filter.

In some embodiments, the at least one filter comprises polyether sulfone.

The filter may have an average pore diameter of at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, suitably at least 5 μm. The filter may have an average pore diameter of at most 10 μm, at most 8 μm, at most 7 μm, at most 6.5 μm, at most 6 μm, at most 5.5 μm, typically at most 5 μm.

The filter may have an average pore diameter of at least 0.2 μm, at least 0.5 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm or at least 1 μm. The filter may have an average pore diameter at most 2 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, or at most 1 μm.

In some embodiments of the described devices, the filter has a flow rate of at least 100, at least 120, at least 140, at least 160, suitably at least 180 ml_/min/cm² of water at 69 kPa. In some embodiments, the filter has a flow rate of at most 300, at most 280, at most 260, at most 240, at most 220, at most 210, at most 200, suitably at most 190 mL/min/cm² of water at 69 kPa.

The device may in any aspect further comprise a source comprising a culture medium suitable for maintaining the viability of the cells. The device may in some embodiments be configured such that after the fluid sample passes through the filter, the filter is contacted with the culture medium. The culture medium may be comprised within a sealed reservoir, and released after the fluid sample has passed through the filter. The devices may further comprise at least one lid, wherein the lid is configured to engage with the reservoir such that the culture medium is released when the lid is applied to the device, and a sealed chamber is created comprising the filter and the culture medium.

In any aspect, at least a portion of the filter may be coated with a protein, suitably wherein the protein is collagen.

In a fourth aspect, there is provided a kit, comprising a receptacle for receiving a biofluid sample, a filtration mechanism comprising at least one filter, the filter comprising polyether sulfone, wherein the filter has an average pore diameter of between about 3 μm and about 6 μm, and a source comprising a culture medium suitable for maintaining the viability of mammalian cells. The receptacle and filtration mechanism are configured to be attached such that a biofluid sample can pass from the receptacle through the at least one filter.

In a fifth aspect, there is provided a kit, comprising a receptacle for receiving a biofluid sample, a filtration mechanism comprising at least one filter, the filter comprising polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate, and a source comprising a culture medium suitable for maintaining the viability of mammalian cells; and at least one lid configured to attach to the filtration mechanism in order to create a sealed chamber comprising the filter and the culture medium. The receptacle is collapsible such that on compression, positive pressure is applied to a received biofluid sample to drive it through the filter.

In a sixth aspect, there is provided a kit, comprising a receptacle for receiving a biofluid sample, a filtration mechanism comprising at least one filter, the filter comprising polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate, a source comprising a culture medium suitable for maintaining the viability of mammalian cells, and at least one lid configured to attach to the filtration mechanism in order to create a sealed chamber comprising the filter and the culture medium. The kit also comprises an air pump configured to attach to the receptacle and apply a positive pressure to the biofluid sample. The air pump may comprise a compressible bladder.

It can be appreciated that the further features of the above-described aspects are not intended to be limited to a specific aspect, and can be applied in many cases to the invention in its other aspects. In particular, the kits as described can be further defined similarly to the devices. BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows a schematic representation of a device according to an embodiment of the invention.

Figure 2 shows devices according to some embodiments of the invention.

Figure 3 shows diagrams of a device according to an embodiment of the invention (similar to that of Figure 2A) and its component parts, as well as images of a device at various stages in its use. 3A shows an exploded version of various component parts; 3B shows a cross-section of an assembled device; 3C and 3D respectively show open and collapsed versions of the collapsible cup; 3E shows cross sections of a device before, during and after use (left to right: open with biofluid in cup; collapsed with biofluid passed through filter / nozzle; ready for shipment with medium in contact with filter and lid secured).

Figure 4 shows the results of experiments investigating the effect of extended urine exposure on cell viability.

Figure 5 shows the results of experiments investigating the effect of centrifugation on cell recovery.

Figure 6A, B and C show the results of experiments investigating the relative efficiency of methods using filter devices according to embodiments of the invention compared to centrifugation methods.

Figure 7 shows the results of an experiment investigating viability of urine-derived renal epithelial cells (URECs) on nitrocellulose filters.

Figure 8 shows a timeline of experiments used to generate data for Figure 6

Figure 9 shows the results of an experiment investigating the proliferation of URECs on different filter materials. Figure 10 shows the results of an experiment investigating the survival of URECs after filtration through or plating onto different filter materials.

Figure 11 shows the results of an experiment investigating the viability of URECs after incubation in urine or culture medium.

DETAILED DESCRIPTION

Unless otherwise indicated, the practice of the present invention employs techniques of chemistry, computer science, statistics, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the comprehension of a person of ordinary skill in the art. Such techniques are also explained in the literature, for example, T. Cormen, C. Leiserson, R. Rivest, 2009, Introduction to Algorithms, 3rd Edition, The MIT Press, Cambridge, MA; L. Eriksson, E. Johansson, N. Kettaneh-Wold, J. Trygg, C. Wikstom, S. Wold, Multi- and Megavariate Data Analysis, Part 1, 2nd Edition, 2006, UMetrics, UMetrics AB, Sweden; M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The inventors have developed a user-friendly filtration device, which captures viable cells from fluid samples such as urine and breast milk, at the point of sample collection and preserves them, increasing their longevity, for example for 24 hours. The device enables collection of live cells from the donors of interest remotely (for example, by mail) for subsequent expansion and analysis of cell lines in laboratory settings.

Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention. All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "comprising" means any of the recited elements are necessarily included and other elements may optionally be included as well. "Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of” means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

The term ‘biofluid’ as used herein refers to a liquid produced by the body which comprises or may comprise body cells. Such biofluids include blood, biopsy fluid, urine, saliva, breast milk, semen, lymph and perspiration. The term is intended to be synonymous with the terms ‘biological fluid’ and ‘body fluid’. In particular, the present invention is especially contemplated for use with body fluids which can be easily and safely obtained outside a clinic or laboratory setting, such as urine or saliva. In specific embodiments of the invention the biofluid comprises urine, or solution that is comprised of an extract or derivative of urine.

The term ‘receptacle’ as used herein refers to an open or closed container or chamber in which the biofluid is collected.

The terms ‘filter, ‘membrane’ and derived terms as used herein refer to size separation means which retain cells present in the biofluid when it passes through these means, while allowing smaller components of the biofluid to pass through. In particular, these terms relate to filter media, such as microfilter membranes, with a porosity sufficient to allow selective retention of the cells comprised in the biofluid. The pore structure of a given filter may be defined e.g. by the median and average diameter of the pores, the pore size distribution, and the porosity of a material. Typically, the properties of the filter medium regarding the median diameter of the pores, the pore size distribution, and the porosity are selected in such a way that the filter medium is suitable for allowing selective retention of the cells comprised in a biofluid sample, such as a urine sample. The invention provides means to firstly separate cells from a biofluid sample, and also to maintain the viability of the cells. This allows the cells to be kept viable for transportation, storage and/or subsequent analytical purposes. Accordingly, in one embodiment the device of the invention comprises a receptacle for receiving the biofluid sample, a filtration mechanism comprising at least one filter; and a source comprising a culture medium suitable for maintaining the viability of the cells. The invention is configured such that after the fluid sample passes through the filter, the filter is contacted with the culture medium.

Figure 1 shows a schematic representation of a device according to an embodiment of the invention.

1. Biofluid sample collection chamber/receptacle, for example 50-500 ml in volume.

2. Waste collection chamber for receiving filtered fluid, again for example 50-500 ml in volume. Waste may in some embodiments be discarded directly, with no waste collection chamber therefore needed.

3. Direction of the transfer of sample from (1) to (2), facilitated by a pressure difference between (1) and (2) and/or by gravitational force, which can be activated/applied by the user upon collection of the sample, or can be passive, for example when gravitational force is used.

4. Cell capture mechanism, containing a filter membrane with properties that enable the capture of cells of interest, and their survival and/or proliferation. This filter membrane also allows components of a size smaller than the desired cells to pass through the filter membrane. These small components include water, salts, proteins, lipids, nucleotides and small organic molecules, and similar.

5. Mechanism that allows the transfer of the membrane/filter containing captured cells from chamber (4) to chamber (6).

6. Optional detachable chamber, containing media that supports growth and proliferation of captured cells for 24-48 hours.

Figures 2 and 3 show further embodiments of devices according to the invention.

The device of the invention operates on the principle of filtration. The fluid sample (which may be urine, or another biofluid) is collected in a receptacle, which may suitably be collapsible, for example a collapsible cup (10) or separable outer funnel (12) and can be made of silicone or other suitable materials, see Figure 3C, D. The illustrated embodiments all include a central hub (20), with embedded membrane (21). Further optional features can include hub adapters (13) and ring holders (14) or similar structures to connect the receptacle to the central hub, and lids (30, 15) to create enclosed environments as appropriate before, during or after use, for example to contain the cells suspended in medium inside the hub for transportation.

It has been determined that the specific gravity of the urine samples used can affect the process, especially where samples are filtered by gravity. Normal physiological range for urine specific gravity is 1.005-1.030. Preferably, the samples used have specific gravity of 1.020 or less. Accordingly, in some embodiments of the methods used herein, specific gravity is adjusted by dilution to be below 1.020.

Once the fluid has been collected, the central hub or filter cap (20), containing a filtration apparatus comprising one or more filters (21) within a chamber, one or more encapsulated media cartridges or reservoirs (23) which comprise a culture medium suitable for maintaining the viability of cells comprised within the fluid sample, and a one-directional valve or nozzle (22) may be screwed on or otherwise attached, see Figure 3.

In some embodiments, the receptacle may already be connected to a filtration apparatus, for example the filter could be connected to the bottom of the receptacle, and filtration can begin through that filtration apparatus. In such cases, after the receptacle has been compressed to aid filtration, as discussed below, a lid could be attached to the receptacle to create a closed environment.

The filtration step involves the fluid being passed from the receptacle through the filter. This may occur by squeezing or otherwise reducing the volume of the collapsible receptacle by mechanical action of the user, such that the fluid is pushed through the filter and the nozzle by positive pressure. In the present context, ‘collapsible’ means that the effective volume of the receptacle can be reduced such that pressure is applied to the contained fluid. In some embodiments, such as shown in Figure 3C and 3D, the collapsible vessel has a tapered shape and a structure such that when pressure is applied it folds into a successively smaller and eventually flattened shape. Alternatively, a receptacle can be collapsible in any other way, such as being made of a flexible material which can be compressed by hand to reduce its volume. Such arrangements can be elastic, such that they tend to resume their original shape after compression, which may be achieved by the use of elastic material or integral springs or other resilient members, or they may retain their reduced volume after compression. A collapsible (or other non-collapsible) receptacle may be provided with a grip or rigid base for ease of applying pressure (11) This may require closing an open receptacle with a lid or other closure mechanism, so that the only exit for the biofluid from the receptacle is through the filter. In some embodiments, fluid may pass through without additional pressure, for example by gravity action, particularly, for example, in the embodiment shown in Figure 2B. The one directional nozzle (22), if used, prevents re-entry of fluid after microfiltration, and allows for maximum collapse of the receptacle, thereby reducing size for ease of transport. Other methods of applying a pressure to the fluid are also considered instead of or in addition to the use of a collapsible receptacle. For example, a pump may be used to apply a positive or negative pressure that drives the biofluid sample through the membrane. Accordingly, an air pump, comprising for example a compressible bladder and configured to connect to the receptacle and form a seal, may be supplied, and may be attached to the receptacle by a user and activated in order to apply a positive pressure. Advantageously, such a pump may be sized and configured to be used by an individual user at home. A plunger may be provided which, when applied to the receptacle, reduces the contained volume similar to the action of a piston or a syringe.

Without wishing to be bound by theory, it is thought that a major factor in the survival of cells is the shear stress that cells are subjected to during the filtration process. It is also thought that filtration using gravity in open configuration (that is, where the fluid being filtered is exposed to atmosphere) is gentlest, and leads to the highest cell yields. The present inventors have determined that an optimal combination of membrane parameters (pore size, membrane diameter and porosity) enable highly successful isolation of cells.

The filter used in these or any embodiment of the invention is suitable for trapping the cells contained in the fluid sample, and for maintaining the viability of these cells. In some embodiments, the filter comprises a polymer. The polymer may be selected from the group consisting of one or more of polycarbonate, polyester, polyether sulfone, stabilised/ regenerated cellulose and polyethylene terephthalate.

Regenerated cellulose is a man-made fibre, obtained by the conversion of natural cellulose into a soluble cellulosic derivative, generally by using chemical dissolution, followed by purification and finally by regeneration into the fibre (Alger 1996). In some embodiments, the polymer may comprise polytetrafluoroethylene. Suitable commercially available membranes include Durapore polyvinylidene fluoride membranes (Merck KGaA), Whatman Polyethersulfone (GE Healthcare), Polyethersulfone microfilter membranes (Sartorius), Hydrosart stabilized cellulose membranes (Sartorius), Polyvinylidene Fluoride (Pall corporation), and Millicell polytetrafluoroethylene (Merck KGaA). Preferably, the filter comprises or consists of polyether sulfone.

The filter may suitably be porous, in particular having an average pore diameter of at least around 0.2 μm, and at most around 2 μm. Typically, the average pore diameter is around 0.8 μm, or is around 1 μm. The average pore diameter may be at least 0.2 μm, at least 0.5 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm or at least 1 μm. The average pore diameter may be at most 2 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, or at most 1 μm. Such pore diameters may be particularly useful in embodiments where mechanical action is used to create a pressure differential in order to pass the biofluid sample through the filter.

In some embodiments, the filter has an average pore diameter of around 5 μm. The average pore diameter may be at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, or at least 5 μm. The average pore diameter may be at most 10 μm, at most 8 μm, at most 7 μm, at most 6.5 μm, at most 6 μm, at most 5.5 μm, or at most 5 μm. In some specific embodiments the upper limit of the pore size is 5 μm. Such pore diameters have been surprisingly determined to allow for efficient flow rate while successfully isolating viable cells, with flow being driven only by gravity, as demonstrated in the Examples. Larger pore diameters risk allowing cells to pass through, while smaller pore diameters may require additional pressure to allow for adequate rates of flow, which may be damaging to the cells for recovery.

Given the above information on average pore diameter, the skilled person will be aware of the degree to which pore diameter can reasonably vary while maintaining the effective properties of a given average pore diameter and enabling a particular flow rate (see below). Of course, while pores significantly smaller than the average are unlikely to much affect flow rate or cell isolation (although they may clog), pores significantly larger than average may allow loss of cells and an abnormally high flow rate.

The present inventors have determined that a particular flow rate is associated with optimum isolation of viable cells from urine. It can be appreciated that flow rate is associated with various properties of a filter to be used, with a number of these properties being discussed elsewhere, such as the filter diameter, filter material, pore size, and porosity (number of pores per unit area). The flow rate therefore varies with these characteristics and others in a manner which may be difficult to predict from a given set of characteristics, but is relatively easy to determine for a given filter.

Similar to the discussion on pore diameter elsewhere herein, the flow rate of filters can determine whether flow can be driven by gravity only, or if additional pressure is required. In some instances, flow by gravity is preferred since the addition of pressure can damage the cells to be recovered. Accordingly, in some embodiments, the flow rate, measured with respect to water in mL/min/cm² at 10 psi (approximately 69 kPa) is at least 100, at least 120, at least 140, at least 160, suitably at least 180 mL/min/cm² at 10 psi. In some embodiments, the flow rate, measured with respect to water in mL/min/cm² at 10 psi (approximately 69 kPa) is at most 300, at most 280, at most 260, at most 240, at most 220, at most 210, at most 200, suitably at most 190 mL/min/cm² at 10 psi. In some specific embodiments, the flow rate is between about 180 and about 190 mL/min/cm² at 10 psi.

Similarly, filter diameter and porosity can be selected in order to allow for a target flow rate. Standard diameters and porosities can accordingly be used, where appropriate. For example, filters of 47mm in diameter can be used.

Under standard protocols, urine is kept refrigerated, typically at 4°C, until it can be centrifuged. The present inventors have found surprisingly that cell suspensions following filtration should be kept at room temperature, as this leads to higher cell recovery, compared to when cell suspensions are kept at 4°C. Therefore, in embodiments of the invention, the urine or other biofluid sample can be maintained at room temperature between being obtained from a subject and being passed through the filter. Similarly, the cell suspensions can be kept at room temperature after filtration and before further processing. In this respect, room temperature can be considered to be between about 15°C and about 25°C, suitably between about 18 °C and about 22 °C.

In some embodiments, for example as shown in Figures 3A and B, after substantially all the fluid has been filtered, and the cup (10) is fully collapsed, the lid (30) is twisted on. The lid (30) and the filter cap (20) are configured in such a way such that when the lid is attached to the filter cap, the encapsulated media cartridge bursts or is otherwise triggered to release the culture media, which then floods the chamber which contains the filter now comprising the cells which have been filtered from the fluid sample, see Figure 3E. The receptacle, lid and cap now create a liquid-tight chamber comprising the filter, the cells trapped thereon, and culture medium to support the cells. The collapsed device then can simply be put into an envelope and shipped to the laboratory with cells remaining viable for at least 24 hours. In other methods, cell preservation medium is added by any convenient means, or cells are washed off the hub and transferred to suitable containers (such as 15 ml Falcon tubes) for transportation.

Two major ways of maintaining the cells retained by filtration are considered. Firstly, cells could be maintained in suspension, floating in the culture media, without adhering to the membrane. This would allow for easy transfer of the cells out of the device once cells arrive at their destination. Secondly, cells can adhere to the surface of the membrane even after contact with the culture media. Recovering the cells from the device would be more challenging in this case. Whether cells remain in suspension or adhere to the membrane depends largely on the membrane material chosen, and on any additional coating of the membrane (for example coating with cellulose or collagen).

Human urine of healthy subjects contains a variety of cells that originate from the kidneys (e.g. podocytes, renal progenitor cells, proximal tubule cells), immune system cells (e.g. leukocytes and macrophages), and urological tract cells (e.g. bladder epithelial cells) (Abedini et al, 2021), tumour cells may also be present (Jiang et al 2019). While the nature of the cells to be isolated will affect the choice of suitable culture media, non-limiting examples which can be used in this and other embodiments of the invention can include REGM Renal Epithelial Cell Growth Medium (Lonza, Basel), or DMEM/F12 (ThermoFisher) supplemented with REGM SingleQuots factors (human epithelial growth factor, Insulin, Hydrocortisone, Transferrin, Triiodothyronine, Epinephrine), glucose, 1- 10% FBS as well as antifungal and antibacterial agents (e.g Penicillin, 100-200 units/ml, Streptomycin (100-200 μg/ml) and Amphotericin B (2.5-5 μg/ml)). The culture medium or media is suitably provided within a sealed cartridge, chamber or reservoir, which advantageously can maintain sterility until the device is used.

In other embodiments, a similar receptacle and filter system as discussed above can be provided. After filtration of the fluid sample, the filter with trapped cells can be removed and placed in a sealable chamber which comprises suitable culture medium, or can have suitable culture medium added to it. Alternatively, in embodiments where the cells do not stay attached to the filter after contact with the culture medium, the cells in the retentate / culture medium can be transferred out of the device into another sealable chamber/container. First, separation of cells from biofluids at the point of collection allows the normalisation of the cell isolation method by substantially removing variability in the time taken to process the sample, which with current techniques is in the range of several hours. Additionally, cell extraction and preservation techniques that do not require specialised laboratory equipment such as centrifuges, means that donors/patients can collect their samples at home, without having to visit a hospital or a laboratory, making cell sourcing of patient- specific cells efficient and scalable, ultimately enabling a shift towards personalised medicine. As demonstrated in the below examples, the described devices and methods achieve improved isolation of viable cells even compared with standard centrifugation methods.

The methods of the invention may be used in the isolation of cells from biofluid samples taken from a range of biological sources for therapeutic, agricultural or veterinary purposes. In particular embodiments the biofluids are obtained from animal subjects, including humans, such as adult or juvenile human subjects (including neonates). Non- human animal subjects may be pets or livestock, such as mammals including primates, cattle, horses, sheep, rats, mice, guinea pigs, rabbits, goats, dogs, and cats.

EXAMPLES

1. Model systems for validation

Assessment of the current 4-hr centrifugation protocol to recover cells from urine, using model systems, determined firstly that extended exposure of cells to urine leads to significant cell losses (Figure 4) and also that centrifugation is not suitable to recover low quantities of cells from large amounts of fluid (Figure 5).

Mouse inner-medullary collecting duct (IMCD3) cells were grown in Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12; Thermo Fisher, 11320033) supplemented with 10% fetal bovine serum (FBS; Gibco, 10500-064) and 1% penicillin- streptomycin (Sigma, P4458), and maintained in a 37°C incubator with 5% CO₂.

For urine exposure studies, cells were trypsinised (Gibco, 25200072), pelleted and resuspended in different media-to-urine ratios, and incubated for 2h or 4h, at 4°C or room temperature. For centrifugation studies, cells were trypsinised, pelleted and resuspended in PBS at different concentrations. Cell suspensions were then centrifuged at 400g for 10min using 50ml falcons, and plated in growth media. A resazurin assay was performed the following day.

Figure 4: Effect of extended urine exposure on cell viability. IMCD3 cells were exposed to pooled human urine from 5 donors, for 2 and 4 hours. Around 65% cells were lost following 2hr exposure, and 90% were lost following 4hr exposure. (n=3, error bars= ±SEM).

These experiments confirmed rapid deterioration of cells from a model cell line (mouse inner medullary collecting duct 3, IMCD3), when exposed to urine, with up to 65% of cells lost after 2 hours of exposure to urine.

To investigate centrifugation as a suitable method to process urine samples with the aim of recovering viable cells, different numbers of IMCD3 cells were suspended in PBS and centrifuged at 400g for 10 minutes. Figure 5 shows the percentage of cells retained after centrifugation.

Figure 5: Effect of centrifugation on cell recovery. Different numbers of IMCD3 cells, suspended in PBS were centrifuged at 400g for 10min, to replicate conventional urine- processing protocol. Over 80% of cells are lost at low concentrations, compared to 25% at higher cell concentration. (n=3, error bars= ±SEM).

These data confirm that more than 80% of cells are lost at low concentrations (300K/100ml), while cell recovery improves with higher cell concentrations (5000K/100ml), when a centrifugation protocol commonly used to process urine samples is followed. This is of particular concern, because intrinsic biochemical properties of the biofluid may affect the likelihood for cells to be recovered, such that it appears that a ‘critical mass’ of particles may be needed for effective recovery to be achieved. This may partially explain the inconsistencies of the results reported in the literature.

These feasibility studies suggest that immediate separation of cells from urine, without using centrifugation, could lead to higher cell yields.

2. Validation of filter devices Experiments were carried out to compare the efficiency of filtration methods and devices as described herein with centrifugation in obtaining viable cells from urine samples.

Filter device

A filter device substantially as described above and as shown in Figure 2B using 3D printing. The filter used was polyethersulfone, with diameter 47mm, and 5 μm pore size (Sterlitech). Following urine filtration (20-100ml), the outer funnel was removed and bottom lid attached. Medium (12ml) was placed inside the hub to resuspend captured cells. Cell suspensions were transferred to 15ml Falcon tubes for transportation.

Cell Culture

Samples processed with filter devices as described above were contrasted with samples produced by centrifugation methods Samples were transferred to 50ml falcons, centrifuged at 1000g for 10 minutes, and the supernatant removed. 10ml of PBS was then added to wash the pellet, followed by the second round of centrifugation at 1000g for 10 minutes. Finally, PBS was removed and 12ml of cell culture medium was added. Cell suspensions were plated across 3 wells in a 12 well plate. Cell culture medium contained DMEM High Glucose/F12 1:1, 1% Penicillin /Streptomycin, 1% amphotericin B, 10% FBS with addition of growth factors). Plates were kept at 37°C incubators (5% CO₂). Half of the media was removed on Day 1 , and 0.5ml of fresh media was added on Days 1 , 2, and 3. Media was replaced completely every other day starting from Day 4. Colonies were quantified manually by two researchers during the second week of culturing, using a 10X microscope.

Results

Two studies were conducted using 63 samples at 4 different sites in London, UK and these found that filter devices as described increase the chances of obtaining viable cells by 26%-30% compared to conventional 2-step centrifugation method (Figure 6A, 6B) and also enrich cell numbers by 80% on average (p-value=0.056) (Figure 6C).

Study 1 (Figure 6A). Forty-four urine samples were collected from patients affected by genetic conditions (Renal tubulopathies (n=18), Bardet-Biedl Syndrome (n=15)) and controls (n=11). Twenty-one were processed in the filter device on site within 30 minutes of collection, while 23 samples were transported to the laboratory and centrifuged within 4 hours. Colonies were quantified 6 to 8 days post-collection using bright-field microscopy. Most of the processed samples formed a variable number of colonies. The distribution of the 3 categories (contaminated, formed colonies, no cells) were different between the two groups of samples: 90.48% of samples processed in a filter device had colonies by day 6- 10 (n=21), while this number in the centrifuged group was lower, 60.87% (n=23). There were samples that showed signs of contamination 1-2 days after plating: approximately 4.76% in the centrifugation group, compared to 21.74% in the cell filtration group. Such samples were discarded.

Study 2 (Figure 6B). Nineteen samples were collected from patients with renal tubulopathies. Each sample was split into two parts: half processed by the filter device, half centrifuged. Colonies were quantified 6 to 8 days post-collection using bright-field microscopy. Overall, 63.16% of samples processed in a filter device formed colonies, compared to 36.84% of samples in the centrifuged category, and the mean number of colonies in the filter device fraction was significantly higher (p-value=0.0098, n=12). Samples where colonies formed in both filter device and centrifuged fractions showed 80% increase in colony number observed, on average (range: from -70% to 233%, n=7).

Figure 6C shows split sample yield differences between filter device and centrifugation fractions as identified in Study 2 above, as also shown in Table 1. Mean number of colonies in the filter device fraction was higher, compared to the centrifuged fraction (n=12, p- value=0.0098) On average, a fraction of the sample processed in a filter device formed 80% more colonies, compared to centrifuged samples (n=7).

Table 1 Mean colonies detected after filtration or centrifugation

3. Validation of further methods

A series of experiments, using expanded human urine-derived cells, also known as urine- derived renal epithelial cells (URECs) confirmed the following:

A. URECs can attach and proliferate on certain filters, with a pore size of up to 1 micron. 4 different materials were tested:

Cellulose Nitrate (CN) Membrane, 0.8 μm pore size (two different suppliers: Sartorius, Goettingen, Germany; Rapid flow Nalgene disposable units (ThermoFisher Scientific, Waltham, USA)

Polycarbonate (PC) Filter (Sterlitech, Kent, USA), 0.2 μm - 1 μm pore size

Polyester (PE) Filter (Sterlitech, Kent, USA), 0.2 μm - 1 μm pore size

Polytetrafluoroethylene (PTFE) Unlaminated Filter (Sterlitech, Kent, USA). 0.2 μm - 1 μm pore size.

Expanded URECs were plated onto filters described above. Due to the physical properties of the membranes, cells could not be visualised by phase contrast microscopy, and instead Resazurin viability assay was performed to determine whether or not cells could attach and grow on the filters tested. This assay is a cell permeable redox indicator used to quantify cellular viability by production of a fluorescent dye (Riss et al 2004). PTFE filters were hydrophobic and did not remain submerged in cell culture media, making them unsuitable for the experiments. Readings from the CN filters were similar to the “blank” negative control, where wells contained no cells, indicating inability of URECs to attach to it, see Figure 7. Reading from the PC and PE filters on the other hand were positive, indicating successful attachment of the cells to filters (data not shown).

B. CoHagen coating of the filters promotes attachment and enhances proliferation of URECs on PC and PE filters (but make no difference for PTFE membranes). A schematic of this experiment is shown in Figure 8. 6 x 10⁴ URECs/well were piated in a 24-well plate (Day 0). Wells contained one of three filter types: PE, PC and PTFE, which were either uncoated or coated with collagen. Control wells contained no filters, and were either coated with collagen or uncoated. All conditions were set up in duplicate or triplicate. Filters were transferred to new wells with media, 24 hours after cells were seeded (Day 1). At Day 2, a 4-hr Resazurin assay was performed to assess UREC viability.

Fluorescence intensity, which is directly proportional to cell numbers, was recorded using a plate reader. The Resazurin assay was repeated 48 hours later (Day 4), and a change in relative fluorescence intensity between the two time points (Day 2 and Day 4) was used to plot the graph. There was a -40% increase in UREC numbers in wells with collagen- coated filters, similar to control wells without filters, while URECs on uncoated PE and PC filters did not proliferate during the 48-hour period, see Figure 9. URECs failed to attach to PTFE filters (data not shown). Error bars are SEM.

Bovine Collagen coating solution (Sigma-Aldrich, St Louis, USA) was used in experiments.

C. URECs do not withstand negative pressure during the filtration process.

Instead of “pushing” cells onto the membrane by positive pressure, as proposed here, negative pressure (i.e. suction/ vacuum to draw fluid through the membrane) could conceivably be applied as an alternative mechanism of filtration. This was tested as shown below.

2.27 x 10⁵ URECs were suspended in 50 ml PBS and filtered using Nalgene Rapid flow Filtration units (ThermoFisher), where original filters were replaced with collagen-coated PC (1 μm pore size), PE (1 μm pore size) and PTFE (1 μm and 0.2 μm pore size) filters. All filters were 47 mm in diameter. These filtration units were then attached by tubes to the suction-generating machines. Filtration was the fastest for the PC filters (6 seconds), followed by PE (75 seconds) and PTFE - 5 minutes. No cells were detected in the filtrate, after centrifuging and plating, confirming that a pore size of 1 μm is suitable for capturing URECs. The filters were then cut out of the filtration units and placed in 6-well plates.

In other wells of these 6 well plates, 2.27 x 10⁵ of URECs were plated. These wells contained collagen-coated PC, PE or PTFE filters, with control wells containing no filters. PTFE filters were hydrophobic and not suitable for cell culture experiments (data not shown).

24 hours after the experiment, a 4-hour Resazurin assay was performed to assess UREC viability across all conditions, and absolute fluorescence intensity, directly proportional to cell number, was used to generate the graph. URECs, captured on PC and PE filters, following the microfiltration process executed via negative pressure as discussed above, were not viable, while plated cells on similar filters (PC plated, PE plated) had the same viability profile as controls.

As shown in Figure 10, this demonstrated that the applied negative pressure was detrimental to the cells, with no URECs surviving the process.

The results of the plated cells also demonstrates that when URECs are plated on filters (i.e. land by gravity) they do attach to and proliferate on the collagen-coated PC and PE filters, but not on CN membranes or unlaminated PTFE filters.

The above indicates that positive pressure is the most suitable mechanism for the proposed device to enable transfer of cells from the fluid sample to the recipient filter.

D. URECs suspended in growth media retain ceil viability

More than 90% of URECs remain viable when suspended in medium for 24hrs (Figure 11). In these experiments 300,000 URECs were incubated in 15ml falcons containing 12ml of medium. 24hrs later, cell suspensions were plated, along with reference numbers corresponding to 100% (300,000 cells) -40% (120,000 cells) of the starting number. A resazurin assay the following day revealed that 92.13% of cells remain viable.

Figure 12 summarises additional experiments, following the same experimental design (300,000 URECs suspended in 12ml of medium inside 15ml falcons). They showed that some cell loss occurs following 48hr incubation, while differences between 4°C and room temperatures (RT) are negligible. Room temperature transportation is more cost-effective compared to 4°C. Effect of agitation on cell viability, expected to occur during shipping is negligible. Agitation in lab settings was modelled by placing falcons containing cell suspensions on horizontal oscillator rotating apparatus. To summarise, over 90% of URECs can be recovered from cell suspensions after 24hr incubation at RT, with agitation. While particular embodiments have been illustrated and described herein, it should be understood that various other changes and modifications may be made without departing from the scope of the claimed subject matter. Moreover, although various aspects of the claimed subject matter have been described herein, such aspects need not be utilised in combination. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the claimed subject matter, which is defined by the appended claims.

References

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5

Claims

1. A method for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types, the method comprising; providing a device comprising a receptacle for receiving the fluid sample, and a filtration mechanism comprising one or more filters, the filter comprising polycarbonate, polyester, polyether sulfone, stabilised/ regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate; placing the fluid sample in the receptacle; passing the fluid sample through the filter by the action of gravity and/or by the application of a positive pressure to the fluid sample; and contacting the filter with a culture medium suitable for supporting cells.

2. The method of claim 1 , wherein the biofluid sample is or comprises urine.

3. The method of claim 1 or claim 2, wherein the biofluid sample is passed through the filter within 30 minutes of being obtained from a subject.

4. The method of any of claims 1 to 3, wherein the biofluid sample is maintained at between 15 and 25°C between its production and being passed through the filter.

5. The method of any of claims 2 to 4, wherein the viable mammalian cells comprise urine-derived renal epithelial cells (URECs)

6. The method of any of claims 2 to 5, wherein the viable mammalian cells comprise immune system cells and/or cells originating from the renal and/or urological tracts.

7. The method of any of claims 1 to 6, wherein the filter has an average pore diameter of at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, suitably at least 5 μm.

8. The method of any of claims 1 to 7, wherein the filter has an average pore diameter of at most 10 μm, at most 8 μm, at most 7 μm, at most 6.5 μm, at most 6 μm, at most 5.5 μm, typically at most 5 μm.

9. The method of any of claims 1 to 8, wherein the fluid sample is passed through the filter by the action of gravity alone.

10. The method of any of claims 1 to 8, wherein the receptacle is compressed to apply positive pressure to the fluid sample.

11. The method of any of claims 1 to 8, wherein an air pump is used to apply positive pressure to the fluid sample, suitably wherein the air pump comprises a compressible bladder.

12. The method of any of claims 1 to 11, wherein the filter comprises a polyether sulfone membrane.

13. The method of claim 12, wherein the filter comprises a polyether sulfone membrane with an average pore diameter of less than about 6 μm.

14. The method of any of claims 1 to 13, wherein the fluid sample is passed through the filter with a flow rate of at least 100, at least 120, at least 140, at least 160, suitably at least 180 mL/min/cm² at 69 kPa.

15. The method of any of claims 1 to 14, wherein the fluid sample is passed through the filter with a flow rate of at most 300, at most 280, at most 260, at most 240, at most 220, at most 210, at most 200, suitably at most 190 mL/min/cm² at 69 kPa.

16. The method of any of claims 1 to 15, wherein after the fluid sample is passed through the filter, the device is sealed to create a liquid-tight chamber.

17. The method of any of claims 1 to 15, wherein after the fluid sample is passed through the filter, the filter is removed and placed within a sealable chamber which comprises a culture medium and/or which is configured to receive a culture medium.

18. A device for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types, comprising: a receptacle for receiving the biofluid sample; a filtration mechanism comprising at least one filter, the filter comprising polyether sulfone; wherein the filter has an average pore diameter of less than about 6 μm.

19. The device of claim 18, wherein the filter has an average pore diameter of greater than about 3 μm.

20. The device of claim 18 or claim 19, wherein the receptacle is collapsible, and is configured such that, on collapsing, positive pressure is applied to the biofluid sample to drive it through the filter.

21. The device of any of claims 18 to 20, further comprising an air pump, wherein the air pump is configured to attach to the receptacle and apply a positive pressure to the biofluid sample, suitably wherein the air pump comprises a compressible bladder.

22. A device for isolating viable mammalian cells from a biofluid sample that may comprise one or more mammalian cell types, comprising: a receptacle for receiving the biofluid sample; a filtration mechanism comprising at least one filter, the filter comprising polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate; wherein the receptacle is collapsible such that on compression, positive pressure is applied to the biofluid sample to drive it through the filter.

23. The device of claim 22, wherein the at least one filter comprises polyether sulfone.

24. The device of ciaim 22 or 23, wherein the filter has an average pore diameter of at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, suitably at least 5 μm.

25. The device of any of claims 22 to 23, wherein the filter has an average pore diameter of at most 10 μm, at most 8 μm, at most 7 μm, at most 6.5 μm, at most 6 μm, at most 5.5 μm, typically at most 5 μm.

26. The device of claim 22 or 23, wherein the filter has an average pore diameter of at least 0.2 μm, at least 0.5 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm or at least 1 μm.

27. The device of any of claims 22, 23 or 26, wherein the filter has an average pore diameter at most 2 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, or at most 1 μm.

28. The device of any of claims 18 to 27, wherein the filter has a flow rate of at least 100, at least 120, at least 140, at least 160, suitably at least 180 mL/min/cm² of water at 69 kPa.

29. The device of any of claims 18 to 28, wherein the filter has a flow rate of at most 300, at most 280, at most 260, at most 240, at most 220, at most 210, at most 200, suitably at most 190 mL/min/cm² of water at 69 kPa.

30. The device of any of claims 18 to 29, wherein the device further comprises a source comprising a culture medium suitable for maintaining the viability of the cells.

31. The device of claim 28, wherein the device is configured such that after the fluid sample passes through the filter, the filter is contacted with the culture medium.

32. The device of claim 28 or 29, wherein the culture medium is comprised within a sealed reservoir, and is released after the fluid sample has passed through the filter.

33. The device of claim 30, further comprising at least one lid, wherein the lid is configured to engage with the reservoir such that the culture medium is released when the lid is applied to the device, and a sealed chamber is created comprising the filter and the culture medium.

34. The device of any of claims 18 to 33, wherein at least a portion of the filter may be coated with a protein.

35. The device of claim 34, wherein the protein is collagen.

36. A kit, comprising: a receptacle for receiving a biofluid sample; a filtration mechanism comprising at least one filter, the filter comprising polyether sulfone, wherein the filter has an average pore diameter of between about 3 μm and about 6 μm; and a source comprising a culture medium suitable for maintaining the viability of mammalian cells; wherein the receptacle and filtration mechanism are configured to be attached such that a biofluid sample can pass from the receptacle through the at least one filter.

37. A kit, comprising: a receptacle for receiving a biofluid sample; a filtration mechanism comprising at least one filter, the filter comprising polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate; a source comprising a culture medium suitable for maintaining the viability of mammalian cells; and at least one lid configured to attach to the filtration mechanism in order to create a sealed chamber comprising the filter and the culture medium; wherein the receptacle is collapsible such that on compression, positive pressure is applied to a received biofluid sample to drive it through the filter.

38. A kit, comprising: a receptacle for receiving a biofluid sample; a filtration mechanism comprising at least one filter, the filter comprising polyester, polycarbonate, polyether sulfone, stabilised/regenerated cellulose, polytetrafluoroethylene and/or polyethylene terephthalate; a source comprising a culture medium suitable for maintaining the viability of mammalian cells; and at least one lid configured to attach to the filtration mechanism in order to create a sealed chamber comprising the filter and the culture medium; and an air pump configured to attach to the receptacle and apply a positive pressure to the biofluid sample, suitably wherein the air pump comprises a compressible bladder.