WO2013182898A1

WO2013182898A1 - Method and kit for obtaining amniotic stem cells from amniotic membrane

Info

Publication number: WO2013182898A1
Application number: PCT/IB2013/001311
Authority: WO
Inventors: Chris Goodman
Original assignee: Virgin Health Bank Qstp-Llc
Priority date: 2012-06-08
Filing date: 2013-06-07
Publication date: 2013-12-12
Also published as: EP2885402A1; GB201210147D0

Abstract

The invention relates to methods and kits for obtaining amniotic stem cells from amniotic membrane by physical separation. Preferred methods of the invention are based on purely physical detachment of the cells. Preferred methods and kits of the invention are free or substantially free of enzymes such as proteases. The invention also relates to cells obtainable by these means and therapeutic uses of these cells.

Description

METHOD AND KIT FOR OBTAINING AMNIOTIC STEM CELLS FROM AMNIOTIC

MEMBRANE

Field of the Invention

The invention relates to methods and kits for obtaining amniotic stem cells, cells obtainable by these means and therapeutic uses of these cells.

Background to the Invention

Amniotic membrane is a tissue of particular interest for obtaining stem cells for use in therapy, compared to other stem cell sources, because amniotic membrane is readily obtained from term placental tissue after parturition. This tissue is highly abundant and is routinely discarded postpartum. No invasive procedures are required to obtain amniotic membrane and amniotic membrane stem cells, unlike the procedures for obtaining stem cells from adult tissues such as bone marrow. Furthermore, use of human amniotic membrane and placental tissue does not involve the destruction of human embryos, and obtaining cells from these tissues thus avoids the controversy and ethical considerations surrounding the use of human embryonic stem (ES) cells (Diaz-Prado ef a/, Differentiation 1 (2011), 162-171).

Amniotic stem cells obtained from amniotic membrane may be used therapeutically, for example in the fields of regenerative medicine, cell therapy and tissue engineering to treat damaged or diseased tissues (Toda ef al, J Pharmacol Sci 105 (2007):215-228). Amniotic membrane- derived cells can differentiate into all three germ layers and have low immunogenicity and antiinflammatory functions. Amniotic stem cells may be particularly beneficial because of the potential immunomodulatory properties of subsets of these cells (Parolini ef al, Stem Cells 26 (2008):300-31 1).

Cells from the amniotic membrane are of foetal origin (Parolini ef al, supra), although they are not embryonic stem cells as they are obtained post-partum from term amniotic membrane. Compared to other tissues of or associated with the placenta, amniotic membrane has several advantages as a source of stem cells. Cells obtained from the chorionic membrane, which is separated from the amniotic membrane in the methods of the present invention, are be more likely to be contaminated with maternal cells, or become contaminated with such cells after in vitro expansion or culture (Soncini ef al, J Tissue Eng Regen Med 1 (2007):296-305). Amniotic membrane should thus be used preferentially to chorionic membrane when isolation of foetal cells is required. Usually, clonal populations of stem cells are desirable, and it is therefore beneficial to isolate cells without maternal contamination. The same considerations apply to stem cells obtained from placenta or umbilical cord blood, which generally contain a variable percentage of haematopoietic and endothelial cells, which may be maternally derived. Amniotic membrane connective tissue is devoid of any vasculature, and such contamination is thus avoided (Alviano et al, BMC Developmental Biology 7 (2007): 11-25).

Current methods for obtaining amniotic stem cells are based on mechanical separation of the amniotic membrane from the underlying chorionic membrane followed by enzymatic digestion to release the stem cells from the amniotic membrane (Diaz-Prado et a/, Differentiation 1 (2011), 162-171 ; Manuelpillai er a/, Placenta 32 (2011 ), S320-325). Enzymes used include trypsin and other digestive enzymes such as dispase and collagenase. Nuclease enzymes may also be used. For example, the method described in WO 2008/1 6992 utilises a solution of trypsin and ethylenediaminetetraacetate (EDTA) to separate the cells. The method described in

EP 1535994 and US 2005/0089513 also require treatment with trypsin to remove amniotic epithelial cells from the membrane. This is followed by protease treatment which may include treatment with papain, collagenase, neutral protease and/or DNase, and potentially further enzymes. The examples in these documents describe a method in which the trypsin treatment is repeated four times. The cells in all of these methods are thus subject to substantial manipulation, particularly enzymatic manipulation.

Given the therapeutic potential and importance of stem cells from amniotic membrane, alternative and improved methods for their isolation are needed, in particular with a view to obtaining preparations of stem cells suitable for therapeutic use. In particular, the development of strategies for good manufacturing practice (GMP)-confonning production of amniotic membrane-derived stem cells is desirable (Parolini et al, Stem Cells Dev 19 (2010): 143-154).

Summary of the Invention

The present invention is based on the finding that amniotic stem cells can be obtained from amniotic membrane by physical means. Methods for obtaining amniotic stem cells from amniotic membrane based on physical detachment of the cells from the membrane have thus been developed and are disclosed herein. The methods of the present invention provide an „ improvement over currently used protocols because they allow the stem cells to be obtained in an inexpensive and rapid method that minimises the manipulation of the cells and the use of reagents and chemicals. In preferred embodiments, the method for obtaining amniotic stem cells from amniotic membrane uses purely physical detachment of the cells.

In a first aspect, the invention provides a method for obtaining amniotic stem cells by physical separation of the cells from an amniotic membrane. In some embodiments, the separation comprises agitation of the amniotic membrane. In certain embodiments, the separation comprises scraping of the amniotic membrane. In some embodiments, the separation step does not comprise enzymatic treatment of the amniotic membrane, such as protease treatment. In preferred embodiments, the obtained stem cell preparation is substantially free of exogenous protease activity, such as trypsin,

collagenase and/or dispase activity. In preferred embodiments, the obtained stem cell preparation is substantially free of papain, collagenase, neutral protease and/or DNase activity. In preferred embodiments, the obtained stem cells conform to good manufacturing practice (GMP).

In some embodiments, the method comprises the following steps:

a) scraping one side or both sides of the amniotic membrane;

b) transferring the cells obtained from (a) to one or more sterile containers; c) pelleting the cells; and

d) resuspending the cell pellet(s) in a buffer.

In some embodiments, the method comprises the additional step of rinsing the scraped amniotic membrane after step (a) and pelleting cells from the rinsate.

Optionally, a sample of the obtained stem cells is removed for testing. Optionally, the method further comprises cryopreservation of the obtained stem cells.

In some embodiments, the method comprises or consists of the following steps:

a) scraping one side or both sides of the amniotic membrane;

b) transferring the cells obtained from (a) to one or more sterile containers; c) rinsing the amniotic membrane to obtain cells resting on the surface of the

membrane and transferring the rinsate to one or more sterile containers of (b); d) pelleting the cells, optionally by centrifugation;

e) discarding the supernatant;

f) resuspending the cell pellet(s) in a buffer;

g) optionally, repeating steps (d) to (f) one or more times; and, optionally h) cryopreserving the obtained stem cells.

In some embodiments, the obtained cells are positive for one or more stem cell markers. In some embodiments, the obtained cells are positive for Oct-4.

The invention provides methods for obtaining amniotic stem cells from an amniotic membrane from a mammal. In preferred embodiments, the amniotic membrane is human. Alternatively, the amniotic membrane may be from a mouse, rat, sheep, pig or cow. In a second aspect, the invention provides a kit for obtaining amniotic stem cells by physical separation of the cells from an amniotic membrane. Kits of the invention comprise a tool for physically separating the cells from the amniotic membrane, a sterile container for the separated cells, and instructions for performing a method according to the invention. In some

embodiments, the tool is a cell scraper. In some embodiments, the kit further comprises a sterile freezing bag and a means for transferring the obtained cells into the sterile freezing bag.

In a third aspect, the invention provides an amniotic stem cell obtainable by any of the methods of the invention.

In a fourth aspect, the invention provides a pharmaceutical composition comprising an amniotic stem cell obtainable by a method of the invention, and a pharmaceutical carrier or excipient.

In yet another aspect, the invention provides an amniotic stem cell obtainable by any of the methods of the invention, for use in therapy. The invention further provides a method of treatment comprising administering to a patient an amniotic stem cell obtainable by any of the methods of the invention:

Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.

Definitions

In order to facilitate the understanding of the present description, the meaning of some terms and expressions in the context of the invention will be explained below. Further definitions will be included throughout the description as necessary.

The term "amniotic stem cell" as used herein shall be taken to mean a stem cell obtained from amniotic membrane. There are several types of amniotic stem cells known in the art

(Manuelpillai ef al, Placenta 32 (2011), S320-325). The term "stem cell" refers to cells that are not terminally differentiated, or are undifferentiated. Stem cells are typically capable of self- renewal and differentiation into one or more mature or differentiated cell types. Monopotent stem cells, also known as "unipotent" or "precursor" cells, can differentiate into one type of cell only (for example hepatocytes). Multipotent stem cells can give rise to different cell types of a single tissue (for example, haematopoietic stem cells give rise to different constituent cells of the blood). Pluripotent stem cells can give rise to cells from any of the three germ layers:

endoderm (interior stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). Pluripotent stem cells can give rise to any foetal or adult cell type. However, they cannot develop into a foetus or adult because they lack the potential to contribute to extraembryonic tissue, such as the placenta. Totipotent stem cells can differentiate into all cell types required to form an embryo, including extraembryonic tissues such as the placenta, and can develop into a foetal or adult animal. The only cell types capable of doing this are embryonic stem cells derived from an embryo at or before the early blastocyst stage. Amniotic stem cells are not "embryonic stem cells" and are not totipotent: human amniotic stem cells are not capable of developing into a human. As described herein, the amniotic membrane, from which the amniotic stem cells are obtained, is expelled after parturition. The term "amniotic membrane" as used herein refers to the inner membrane of the amniotic sac, which faces the amniotic cavity and the amniotic fluid. The amniotic membrane is sometimes referred to as the "amnion". The amniotic membrane is distinct from the chorion (also known as the chorionic membrane), which is the outer membrane of the amniotic sac. The amniotic membrane is separated from the chorion by a layer of loosely arranged collagen fibres. The amniotic membrane is also distinct from other locations from which stem cells may be obtained, for example the amniotic fluid or umbilical cord blood. Amniotic membranes used with the present invention are obtained from placenta from a mammal, preferably a human, and are obtained after parturition. The term "physical separation" as used herein refers to the separation or dissociation of cells from the amniotic membrane by a physical action, i.e. a non-chemical and non-biological (e.g. enzymatic) action. The separation or dissociation is achieved by the physical action and not by chemical or biological action. The separation is achieved by physical means even in methods of the invention where chemical or biological agents are included in one or more steps of the method. Physical separation includes separation by any mechanical action or movement, either manual or automated, including but not limited to agitation, aspiration, scraping and/or application of shearing forces to the surface of the membrane.

The term "mechanical" as used herein refers to separation caused by movement, physical forces, properties or agents, for example separation caused by shearing forces, suction, friction or abrasion.

The term "manual" as used herein refers to the use of physical, non-automated action carried out by hand i.e. by the person carrying out the method (the "operator").

The term "automated" as used herein refers to physical action carried out by an apparatus, such as a machine, which requires limited or no direct action from an operator i.e. which does not require constant monitoring or action by the person carrying out the method. The term "agitation" as used herein refers to vigorous movement sufficient to separate or dissociate stem cells from the amniotic membrane. Agitation may be carried out by any method known in the art, including but not limited to use of shaking apparatus.

The term "aspiration" as used herein refers to the application of suction sufficient to dissociate stem cells from the amniotic membrane. Aspiration may be carried out by any method known in the art, including but not limited to the use of micropipette aspiration or fine needle aspiration. The term "scraping" as used herein is to be given its usual meaning and refers to the drawing of an appropriate tool across the surface of the amniotic membrane, such that the tool is in contact with the membrane but does not penetrate the membrane, while applying pressure sufficient to separate the stem cells from the membrane. The term "cell scraper" refers to any tool suitable for scraping cells, and should be sterile.

Suitable tools are described more fully below.

The term "buffer" as used herein is to be given its usual meaning and refers to a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH. Buffers used with the present invention include any buffer suitable for handling or maintaining cells, including any buffered solution with ionic concentrations suitable for use with eukaryotic, typically mammalian, cells. Buffers for use with the invention include eukaryotic cell culture media and other buffered solutions, and are discussed further below. The term "enzymatic treatment" as used herein refers to any treatment of the membrane with an enzyme, typically an enzyme that may be used to separate cells. Typically, such enzymes will be proteases, including but not limited to trypsin, collagenase and dispase. However, in some embodiments of the invention it may also be beneficial to avoid treatment with other enzymes, such as nucleases. It may be beneficial to avoid treatment with other enzymes, including but not limited to papain, collagenase, neutral protease and/or DNase.

The term "protease activity" as used herein refers to the presence of an active protease that can be used to separate amniotic stem cells from the amniotic membrane, for example by disrupting intercellular connections or breaking down extracellular matrix. The term "exogenous" means that the protease is not produced by the obtained amniotic stem cells and is not endogenous to a preparation of unmanipulated amniotic stem cells. Where an "exogenous" protease is used, it is added to the amniotic membrane and/or stem cells during the separation method and/or added to the stem cells or cell preparation obtained by the method. The term "substantially free of a substance as used herein means a non-significant amount of the substance, for example similar to or the same as the amount in a negative control, or at or around background levels. The term "substantially free of includes "completely free of.

As used herein, the term "significant expression" or its equivalent terms "positive" and "+" when used in regard to a cell marker shall be taken to mean that, in a cell population, more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 98%, 99% or even all of the cells express said marker.

As used herein, "negative" or "-" as used with respect to cell markers shall be taken to mean that mean that, in a cell population, less than 20%, 10%, preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % or none of the cells express said marker. As used herein, the terms "treat", "treatment", "treating" and "therapy" when used directly in reference to a patient or subject shall be taken to mean the amelioration of one or more symptoms associated with a disorder, or the prevention or prophylaxis of a disorder or one or more symptoms associated with a disorder. The disorders to be treated include, but are not limited to, a degenerative disorder, a disorder involving tissue destruction, a neoplastic disorder, an inflammatory disorder, an autoimmune disease or an immunologically mediated disease including rejection of transplanted organs and tissues. Amelioration or prevention of symptoms results from the administration of the cells of the invention or cells obtained according to methods of the invention, or of a pharmaceutical composition comprising these cells, to a subject in need of said treatment.

The term "allogeneic" as used herein shall be taken to mean from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. The term "autologous" as used herein shall be taken to mean from the same individual.

The term "immunomodulatory" refers to the inhibition or reduction of one or more biological activities of the immune system and includes, but is not limited to, downregulation of immune responses and inflammatory states as well as changes in cytokine profile, cytotoxic activity and antibody production. 1

8

Detailed Description of the Invention

The present inventors have surprisingly found that stem cells can be readily obtained from the amniotic membrane by physically removing the cells from the membrane. The cells obtained by this method are useful in therapy and research.

Methods

In a first aspect, the invention provides a method for obtaining amniotic stem cells by physical separation of the cells from an amniotic membrane. In preferred embodiments, the invention provides a method for obtaining amniotic stem cells by purely physical separation of the cells from an amniotic membrane. Methods of the invention provide useful amniotic stem cells.

Obtaining the cells by physical separation according to the methods of the present invention has several advantages over the prior art, and particularly over methods requiring enzymatic digestion. The obtained cells have been subjected to minimal manipulation and may be kept free of, substantially free of or are uncontaminated by chemicals or biological molecules (such as enzymes) that may be undesirable in the final population or preparation of cells. In particular, it may be beneficial to avoid certain contaminants in cell preparations for use in therapy.

Methods of the invention are simple, easy to carry out, inexpensive and rapid. For example, methods of the invention are typically more rapid than the methods described in EP 1535994 and US 2005/0089513, which involve four 15-minute incubations with trypsin. Where enzymes (and/or chemicals) are used in the known methods of obtaining amniotic stem cells, each of these components must be rigorously tested to ensure that it conforms to good manufacturing practice, as described in Parolini et al, Stem Cells Dev 19 (2010):143-154. These requirements carry associated expense and regulatory concerns, and significant time and effort is required to set up a validated method. Methods of the invention remove the requirement for enzymes, such as proteases, to separate the stem cells form the amniotic membrane. Stem cells obtained by the physical methods of the invention may also require less purification and/or decontamination from reagents used in the method compared to methods that require chemical or enzymatic (biological) separation of the stem cells form the membrane.

It was unexpected that cells could be obtained from amniotic membrane by physical separation because the known methods use enzymes to carry out the separation step (Diaz-Prado et al, Differentiation ! (2011 ), 162-171). See also WO 2008/146992, EP 1535994 and US

2005/0089513 (described above). The amniotic membrane

The starting material for the methods of the invention is amniotic membrane. Amniotic membranes used in the invention are obtained from placental tissue from a mammal, preferably a human, obtained after parturition (either by caesarean section or by vaginal birth). Methods according to the invention therefore relate to obtaining amniotic stem cells from ex vivo amniotic membrane, and more specifically to amniotic membrane obtained from placental tissue that has been expelled after birth and is therefore no longer required by the foetus. Use of such tissue from a human is generally considered to be ethically acceptable because its use has no impact on the mother or foetus, and this tissue is usually discarded. Placental tissue and amniotic membrane is obtained with maternal consent.

The amniotic membrane is a thin, avascular membrane composed of an epithelial layer and an outer layer of connective tissue. The amniotic epithelium is an uninterrupted, single layer of ectodermally-derived flat, cuboidal and columnar epithelial cells in contact with amniotic fluid in the amniotic cavity. The amniotic epithelium is attached to a basal lamina (also referred to as the "basement membrane") that is in turn connected to the amniotic mesoderm. In the amniotic mesoderm closest to the epithelium, an acellular compact layer is distinguishable, composed of collagens I and III and fibronectin. In the amniotic mesoderm further from the epithelium, a network of dispersed fibroblast-like mesenchymal cells and some macrophages are observed. The amniotic mesoderm is separated from the chorionic mesoderm of the chorionic membrane by a spongy layer of loosely arranged collagen fibres.

At least two populations of stem cells are known to be obtainable from amniotic membrane (Manuelpillai et al, Placenta 32 (201 1), S320-325; Diaz-Prado er a/, Differentiation 1 (2011 ), 162-171). Amniotic epithelial cells (AECs) are found in the amniotic epithelium. Amniotic mesenchymal stromal cells (AMSCs) are found in the amniotic mesoderm. Both cell types are described more fully below under "Amniotic stem cells". Amniotic epithelial cells are obtainable by methods of the invention. Amniotic mesenchymal stromal cells are obtainable by methods of the invention. A mixed population of AECs and AMSCs is also obtainable by the methods of the present invention.

The amniotic membrane will usually be obtained from the placenta by detachment from the chorionic membrane, after separation of the amniotic sac from the placenta. Detachment of the amniotic membrane from the chorionic membrane is typically carried out by mechanical separation, for example by blunt dissection, but any suitable method may be used. For example, the amniotic membrane may be separated by peeling from the chorionic membrane layer, as described in EP 1535994. The separation of the amniotic membrane from the chorionic membrane alone does not result in isolating amniotic membrane stem cells according to the invention.

The amniotic membrane is typically washed after detachment from the chorionic membrane. The membrane may be washed one or more times, for example two, three, four, five, six, seven, eight, nine, ten or more times. The membrane may be washed in any suitable buffer, for example in PBS, a balanced salt solution such as HBSS, or in other suitable solutions, such as but not limited to dilute NaCI solution (for example, 0.9% (w/v) NaCI solution). In some embodiments of the methods of the invention, the placenta is cold stored before taking the amniotic membrane from it and carrying out the method of the invention. In these embodiments, the placenta may be cold stored for up to 4 days after parturition. For example, the amniotic membrane may be stored for up to and including 4 days, 3 days, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1.25 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes or 30 minutes after parturition.

In other embodiments, the placenta is not cold stored prior to separating the amniotic membrane. Where the placenta and/or amniotic membrane are not cold-stored, it is preferable to obtain the amniotic membrane and commence the method of the invention up to a maximum of 48 hours after parturition. For example, the method of the invention may be commenced up to and including 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1.25 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes or 30 minutes after parturition.

Physical separation

The physical separation can comprise agitation of the amniotic membrane. In some

embodiments, shearing forces are applied to the amniotic membrane. Agitation and/or application of shearing forces may be manual or automated, but in all embodiments the forces applied should be sufficient to cause separation of cells from the membrane. In some embodiments, the shearing forces may be applied by forcing air or other gas such as C0₂ or a liquid such as a buffer over the amniotic membrane (generally referred to as "forced air").

The physical separation can comprise aspiration. In such embodiments, suction (reduced air pressure) is applied to the membrane at a level sufficient to dissociate stem cells from the amniotic membrane. Aspiration may be carried out by any method known in the art, including but not limited to the use of micropipette aspiration or fine needle aspiration. Aspiration may be carried out in combination with any of the other physical separation methods disclosed herein. For example, aspiration and scraping can be combined.

The physical separation can comprise scraping. The scraping action is sufficient to detach cells from the membrane. Preferably, the pressure applied to the membrane is the minimum pressure required to detach and obtain cells. Minimising the pressure applied to the membrane reduces the risk of damage to the cells. As a general guide, the pressure applied should not be so great as to regularly tear the membrane, although occasional tearing of the membrane may occur. By "occasional tearing" it is meant that the membrane may tear in up to 10 places, for example in 1-10 places, 3-8 places, 4-5 places or 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 places. A tear may be up to 10 cm in length, for example up to 9, 8, 7, 6, 5, 4, 3, 1 cm in length. .

The optimal pressure can be ascertained via a pilot experiment where an amniotic membrane is cut into pieces or strips, and cells are obtained from each piece or strip. The same method of the invention is used to obtain cells from each piece or strip, but the amount of pressure applied to each one is varied. The obtained cells can then be compared by assessing viability, cell yield and/or further parameters of interest, and the optimal pressure determined. Assays for assessing such parameters will be apparent to one skilled in the art, and exemplary assays are discussed below under "Assays for testing cells".

In some embodiments, scraping is carried out using a cell scraper. The term "cell scraper" refers to any tool suitable for scraping cells. A cell scraper may be a slide glass. Cell scrapers should be sterile. The part of the cell scraper which contacts the amniotic membrane should not be sharp, i.e. a cell scraper should not pierce the membrane during normal operation, although accidental piercing may occur. Cell scrapers for manual use {i.e. user-operated) should comprise a section which contacts the cells ("scraping section"), and a further section or handle which can be held by the user such that the user does not need to contact or touch the scraping section. Optionally, scraping may be carried out using an automated cell scraper. This may take any form, and may be multiplexed i.e. allow the parallel scraping of more than one amniotic membrane sample. Any automated cell scraping apparatus may be used, without limitation. For example, the apparatus and scraping method disclosed in WO 2008/034868 (US

201 1/0124037) may be adapted for obtaining cells from amniotic membrane.

Scraping may also be carried out by adding inert particles to the membrane such that they lie on its surface, then subjecting the membrane to vigorous agitation such that the inert particles abrade the surface of the membrane. The inert particles may be made of glass, or of any other suitable inert material. The term "inert" as used herein means that the particles do not react with any component of the membrane and do not break down chemically. The inert particles should also be substantially smooth. While they need not be completely smooth, they should not have sharp edges which could tear the amniotic membrane or damage the cells. In one embodiment, the inert particles are glass beads.

Any combination of the physical separation methods disclosed herein may be used with the methods of the invention. In some embodiments, the method comprises, consists of, or consists essentially of the following steps:

a) scraping one side or both sides of the amniotic membrane;

d) resuspending the cell pellet(s) in a buffer.

The scraping and/or transferring steps are optionally carried out in the presence of a suitable buffer, facilitating the transfer of the cells to the sterile container in the form of a cell suspension. Any suitable buffer may be used, as described below under "Buffers".

Similarly, any suitable buffer may be used for resuspension of the cells. Resuspension of the cells may be carried out by any suitable method known in the art, for example by drawing the cells through a pipette tip several times until they are evenly suspended in the buffer, and/or by agitation of the buffer. Resuspension of the cells is preferably carried out gently so as to minimise any damage to the cells. Suitable methods for resuspension will be apparent to one skilled in the art.

The method may comprise the additional step of rinsing the scraped amniotic membrane after step (a) and pelleting cells from the rinsate. Rinsing may be carried out using any suitable buffer, as described herein. Rinsing of the membrane may be carried out one or more times. Rinsing allows the recovery of additional cells which may remain on or associated with the amniotic membrane after scraping.

A sample of the obtained stem cells may be removed for testing. Samples may be taken at any stage during the method, and different samples taken at different stages or steps of the method may be compared to assess the changes in properties of the cell as the method is carried out. Assays for testing the cells and assessing properties of interest are described below under "Assays for testing cells". Without limitation, tests may assess viability, total nucleated cell (TNC) count, colony forming units (CFU), cell marker expression, genetic stability, virology and/or microbiology.

Methods of the invention may further comprise cryopreservation of the obtained stem cells. In a multi-step method, this will be done after the final step of the method. Cryopreservation may be carried out by any method known in the art, including but not limited to slow freezing of the cells, usually in the presence of a cryoprotectant. One useful cryoprotectant is DMSO. Slow freezing may be carried out using a programmable temperature-decreasing container and/or a controlled-rate freezer. Cryopreservation may also be carried out by rapid freezing, for example by vitrification using a high cryoprotectant concentration and not requiring a programmable temperature-decreasing container (Moon et a/, Hum Reprod 23 (2008)1760-1770). Preferably, the cryopreservation method minimises ice crystal formation and toxic and osmotic damage to cells. Preferably, the cryopreservation method used is reliable and effective for long-term cell preservation, such that cells show retention of cell surface marker expression and differentiation potential after thawing.

Methods of the invention may further comprise expansion of the obtained stem cells. In a multi- step method, this will be done after the final step of the method. Cell expansion may be carried out by any method known in the art, including but not limited to the methods described below under "Cell culture".

Methods of the invention may comprise or consist of the following steps:

a) scraping one side or both sides of the amniotic membrane;

e) discarding the supernatant;

f) resuspending the cell pellet(s) in a buffer;

9) optionally, repeating steps (d) to (f) one or more times; and, optionally h) cryopreserving the obtained stem cells.

The repetition of steps (d) to (f) can facilitate the removal of non-cellular components derived from any previous steps. As such, this optional repetition can improve the purity of or reduce the level of any contaminants in the final cell preparation. Absence of enzymes

The invention advantageously separates the amniotic stem cells form the amniotic membrane by physical means, such as scraping. Although enzymes or chemical agents can optionally be used in addition, chemical or biological agents are not required to separate the stem cells form the membrane. Therefore, in one embodiment, the separation step is carried out in the absence of enzymes that are used to separate the stem cells from the membrane.

In some embodiments of the methods of the invention, stem cells are separated from the amniotic membrane without enzymatic treatment, such as protease treatment. Protease treatment could include treatment with trypsin, collagenase and/or dispase, but is not limited to these enzymes. Non-protease enzymes may also preferably be omitted from the separation step, for example nucleases. For example, papain, collagenase, neutral protease and/or DNase may preferably be omitted from the separation step. It is beneficial to carry out separation of stem cells from amniotic membrane without enzymatic treatment. This allows the possibility of carrying out methods of the invention without any added or exogenous enzyme. Optionally, one or more steps of the method are performed in the absence of added or exogenous enzyme. Optionally, all of the steps of the method are performed in the absence of added or exogenous enzyme.

The absence of exogenous enzymes in the method provides obtained stem cell preparations that may be substantially free, or free, of exogenous protease activity, such as trypsin, collagenase and/or dispase activity. In reference to protease activity, "substantially free" means that the activity of a protease of interest is close to the lower limit of detection, or close to background levels of activity, using a standard assay for activity of said protease. For example, kits are provided for testing protease activity, such as the Pierce Quantitative Protease Assay Kits produced by Thermo Fisher Scientific. A suitable negative control for determination of "background" levels would be a sample of the cells to which no protease has been added at any stage. Alternatively, a suitable negative control may be provided in a kit for testing protease activity. In reference to protease activity, "free" means that the activity of a protease of interest is below the limit of detection, or at or below background levels of activity, using a standard assay as described above.

Methods of the invention are preferably carried out in accordance with good manufacturing practice (GMP). Preferably, the obtained stem cells conform to GMP. Principles and guidelines of GMP for human cells and tissues are set out by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). For example, relevant guidelines are set out in the EU Cells and Tissues Directive (2004/23/EC), related technical directives 2006/17/EC and 2006/86/EC. Relevant regulations are set out in EU published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products (ATMP). In this regulation, cells and tissues are considered "engineered" if they have been subjected to "substantial manipulation", such as cell expansion, selection or functional activation. The following actions are not considered

"substantial manipulation": cutting, grinding, shaping, centrifugation, soaking in antibiotic or antimicrobial solutions, sterilization, irradiation, cell separation, concentration or purification, filtering, lyophilization, freezing, cryopreservation, and vitrification. In preferred embodiments of the methods of the invention, the obtained cells are not substantially manipulated, wherein substantially manipulated has the sense outlined in the EU regulations discussed above. In preferred embodiments of the methods of the invention, the obtained cells are minimally manipulated. Processing of cells is considered "minimal manipulation" when there is no alteration of the relevant biological characteristics of the cells (US Code of Federal Regulations, 21CFR1271.3). Straightforward cell isolation, separation and cryopreservation can at first analysis be considered as minimal manipulations. When processing alters any biological characteristics and thus potentially alters cell function or integrity, then it is considered "more than minimal manipulation". This latter category must be applied when adequate information does not exist to determine if the processing will alter cell characteristics or not (Pessina et al, Crit Rev Microbiol 34 (2008): 1- 2). Regulatory aspects, including the relevant US regulations for amniotic membrane-derived stem cells (such as 21 C.F.R. § 1271), are discussed further in Parolini et al, Stem Cells Dev 19 (2010): 143- 154, incorporated herein by reference.

The obtained stem cell preparation is typically substantially free or free of exogenous proteases, for example trypsin, collagenase and/or dispase. In reference to proteases, "substantially free" means that the protease of interest is at a level that is close to the lower limit of detection, or close to background levels, using a standard assay for detection of said protease. Assays for detection of a protein of interest, such as a protease, include but are not limited to western blotting and ELISA. A suitable negative control for determination of "background" levels would be a sample of the cells to which no protease has been added at any stage. In reference to proteases, "free" means that the protease of interest is below the level of detection, or at or below background levels, when tested using a standard assay as described above. The obtained stem cell preparation may be substantially free or free of exogenous enzymes including proteases as described above. The obtained stem cell preparation may be

substantially free or free of exogenous enzymes including, but not limited to, trypsin, collagenase, dispase, papain, collagenase, neutral protease and/or DNase.

Where trypsin is present in the methods or stem cell preparations of the invention, its concentration is preferably less than 0.5% (w/v), 0.25% (w/v) or 0.125% (w/v), more preferably less than 0.025% (w/v), still more preferably less than 0.02%, 0.01%, 0.005%, 0.0025%, 0.001%, 0.0005% or 0.0001% (w/v).

Inactivation of trypsin is often achieved using culture media containing foetal bovine serum (FBS). In some methods of the invention, trypsin is not used and FBS can therefore be omitted.

Buffers

Since enzymes are not required in the methods of the invention, and it is possible to avoid the use of enzymes, simple buffers such as PBS can optionally be used in the methods of the invention. Any suitable buffer or combination of buffers may be used with the invention, including but not limited to phosphate-based buffers such as PBS, HEPES-based buffers, sodium bicarbonate-based buffers and balanced salt solution such as HBSS and EBSS.

In some methods of the invention, the buffer used is suboptimal or not suitable for activity of enzymes including but not limited to trypsin, collagenase and/or dispase.

Eukaryotic cell culture media are also useful buffers that can be used in the present invention. Suitable culture media include, but are not limited to, DMEM, MEM and RPMI. Cell culture media for use with the invention are preferably free or substantially free of foetal bovine serum (FBS). Where FBS is used in accordance with the methods of the invention, it is preferably from a country certified to be free of bovine spongiform encephalopathy, in keeping with

recommended practice for clinical stem cell use (Halme and Kessler, NEJM 355 (2006): 1730- 1735).

A particularly preferred buffer for use in the methods of the invention is DMEM. Another particularly preferred buffer for use in the methods of the invention is PBS. A further particularly preferred buffer for use in the methods of the invention is MEM. Combinations of these buffers may also be used.

In preferred embodiments, buffers used with the invention meet good manufacturing practice (GMP) quality or standards for handling stem cells (Halme and Kessler, NEJM 355 (2006): 1730- 1735; Parolini et al, Stem Cells Dev 19 (2010):143-154). Preferably, and particularly where the obtained cells will be used in therapeutic or clinical applications, buffers will be substantially free or free of animal substances. Where animal substances are used, they are preferably from the same organism as the obtained stem cells. For example, where the obtained stem cells are from a human amniotic membrane, any animal products will preferably be from a human. For example, where serum is required, human serum may be used. Where foetal bovine serum is used, it should not exceed 5% (v/v) and should meet the relevant GMP standards. Temperature

Methods of the invention may be carried out at room temperature, for example at about 15?C to 30°C. In some embodiments, the methods of the invention are carried out at or below 15°C, for example at or below 10°C, at or below 5°C, 4°C, 3°C, 2°C or 1°C. In some embodiments of the methods of the invention, for example where enzymatic digestion is avoided, it may be beneficial to carry out certain steps of the method or the whole method at a temperature which is suboptimal for enzyme activity, e.g. at or below 15°C, at or below 10°C, at or below 5°C, 4°C, 3°C, 2°C or 1°C. Cell culture

Methods of the invention may or may not comprise a step of culturing the stem cells, thus allowing them to proliferate, but this step is not required. In some embodiments of the invention, the cells are not cultured. Culturing may also be referred to as "passaging" the cells, or as "subculture". Benefits of excluding a cell culture step include the fact that amniotic stem cells may be difficult to culture, and that it may be difficult to achieve clonal colony formation when cells are passaged in culture (Manuelpillai er a/, Placenta 32 (2011 ), S320-325; Bilic er a/, Cell Transplant 17 (2008):955-968). "Clonal colony formation", also referred to as

"clonogenicity", refers to the ability of a stem cell to self-renew or replicate itself and form a colony of genetically identical cells. In vitro culture may also result in altered phenotype or differentiation potential of the stem cells (Parolini et al, Stem Cells Dev 19 (2010): 143-154).

In some embodiments of the invention, the cells are cultured. Where cells are cultured, this is preferably for no more than 15 passages, more preferably no more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 passages. Culturing allows expansion of the cell population, where this is required; for example, expansion in vitro may be required for some therapeutic applications of the obtained cells. In other therapeutic applications, it may be sufficient to use a smaller number of cells which have not been cultured in vitro. These cells may then expand in vivo after they have been administered to a patient or subject. Methods of the invention may thus comprise a step of incubating the obtained stem cells in a suitable cell culture medium, typically on a solid surface under conditions which allow cells to adhere to the solid surface and proliferate. The material may be a plastic treated to promote the adhesion of mammalian cells to its surface, for example commercially available polystyrene plates, optionally coated with poly-D-lysine or other reagents. Suitable conditions for cell culture will be apparent to one skilled in the art.

Where a cell culture step is included in the methods of the invention, this will typically be carried out after the stem cells have been separated from the amniotic membrane. However, it is possible to culture the cells while they are still attached to the amniotic membrane, i.e. cell culture can be performed in situ on the amniotic membrane. However, this is not preferred.

Kits

As described above, it is beneficial to obtain cells from the placenta soon after parturition, or after cold storage. In all cases, it is beneficial to minimise the time for which the amniotic membrane and obtained cells are handled in carrying out the methods of the invention. Kits of the invention bring together the components required to carry out the methods of the invention, and instructions for carrying out the method, allowing the method to be carried out quickly and reducing the time for which the amniotic membrane and obtained stem cells are handled.

Kits of the invention comprise a tool for physically separating the cells from the amniotic membrane, a sterile container for the separated cells, and instructions for performing a method according to the invention. In some embodiments, the tool is a cell scraper. In some embodiments, the kit further comprises a sterile tray on which the amniotic membrane is placed before performing the physical separation step. In some embodiments, the kit further comprises a sterile freezing bag and a means for transferring the obtained cells into the sterile freezing bag. In some embodiments, the cells are transferred into the sterile freezing bag by injection. In such cases, the means for transferring the cells may be a syringe, optionally with a needle attached. Preferably, the gauge number, which defines the outer diameter of the needle, is 20 or lower, more preferably 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 or lower. In a preferred embodiment, the gauge number of the needle is 16.

Kits of the invention typically do not contain enzymes suitable for separating amniotic stem cells from the amnion.

Good manufacturing practice requires that preparation of a product follows validated standard operating procedures (SOPs). In kits of the invention, the quality of the various components can be tested, standardised and validated, and the kits thus allow methods of the invention to be carried out according to GMP standards.

Kits of the invention may preferably contain components that are substantially free or free of enzymes, in particular proteases. For example, kits of the invention may be substantially free or free of enzymes such as trypsin, collagenase, dispase, papain, collagenase, neutral protease and/or DNase, The kits of the invention preferably do not contain these enzymes. In a particularly preferred embodiment, kits of the invention may be certified to be free of such enzymes. Amniotic stem cells

The invention provides an amniotic stem cell obtainable or obtained by a method of the invention. Preferably, amniotic stem cells of the invention are directly obtained by any of the methods of the invention, i.e. without additional steps after the cells have been obtained.

As described above, cells from the amniotic membrane are usually of foetal origin, and cells of the invention are therefore usually of foetal origin. Cells are foetal in origin due to the fact that the amniotic membrane is one of the extraembryonic tissues developing from the totipotent embryonic stem cells during the early stages of embryogenesis. However, the term "foetal origin" simply indicates that the obtained cells have foetal, rather than maternal, genetic information; "foetal origin" does not mean that the obtained stem cells are embryonic stem cells. As stated above, the amniotic stem cells of the invention are obtained from term placental tissue after parturition, and the stem cells in this tissue are not embryonic stem cells. The amniotic stem cells of the invention are pluripotent, not totipotent, and are not capable of developing into an embryo.

Preferably, cells of the invention are substantially free of maternal contamination. Foetal origin can be verified or tested using methods sensitive enough to detect maternal contamination of 1% or less (Parolini et al, Stem Cells 26 (2008): 300-311). "Substantially free of maternal contamination" means that the cell preparation contains no more than 10% maternal cells, preferably no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% maternal cells, more preferably no more than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.025%, 0.01% or less. In a more preferred embodiment, cells of the invention are free of maternal contamination, meaning that maternal contamination is undetectable or at background levels using a standard assay.

As described above, stem cells from amniotic membrane are known in the art (Manuelpillai et al, Placenta 32 (2011), S320-325). Two types of stem cell are known to be obtainable from amniotic membrane: amniotic mesenchymal stromal cells, and amniotic epithelial cells.

However, it is possible that other useful cell types are obtainable from amniotic membrane (Parolini ef al, Stem Cells 26 (2008):300-311).

As used herein the term "amniotic mesenchymal stromal cell" (also referred to herein as

"AMSC" and sometimes as "amniotic mesenchymal stem cell") shall be taken to mean a cell derived from the extraembryonic amniotic mesoderm. Amniotic mesenchymal stromal cells are capable of giving rise to multiple different types of cell, including cells of mesenchymal lineage, for example osteogenic, chondrogenic and adipogenic cells. Thus amniotic mesenchymal stromal cells can differentiate into various cell types including bone, cartilage, fat cells and fibroblasts. Amniotic mesenchymal stromal cells can also give rise to cells of other mesodermal lineages, for example myocytes (smooth and/or skeletal muscle cells), cardiomyocytes and endothelial cells. Amniotic mesenchymal stromal cells may be capable of differentiating into cell types from all three germ layers. Amniotic mesenchymal stromal cells may be able to differentiate into cells of ectodermal lineage, for example neural cells, and of endodermal lineage, for example pancreatic and/or hepatic cells. The cell types into which AMSCs differentiate may be dependent on various factors, including culture conditions (Manuelpillai ef a/, Placenta 32 (2011), S320-325). Amniotic mesenchymal stromal cells express stem cell markers, including Oct-4. Amniotic mesenchymal stromal cells may also express a variety of other markers, although the levels and pattern of marker expression may vary depending on how the cells are treated and whether or not the cells are expanded in culture. A summary of the markers typically expressed by undifferentiated human amniotic mesenchymal stromal cells is shown in Table 1 :

Table 1 - marker expression by undifferentiated hAMSCs (adapted from Manuelpillai et al, Placenta 32 (2011), S320-325). As used herein the term "amniotic epithelial cell" (also referred to herein as "AEC") shall be taken to mean a cell derived from the amniotic epithelium, i.e. the monolayer of flat, cuboidal and columnar epithelial cells found on the inner surface of the amniotic membrane, in direct contact with amniotic fluid. Amniotic epithelial cells originate from the extraembryonic ectoderm. Amniotic epithelial cells are pluripotent stem cells and have the ability to differentiate into cell types from all three germ layers. Amniotic epithelial cells have the ability to differentiate into cells of ectodermal lineage, for example neural cells. Amniotic epithelial cells have the ability to differentiate into cells of endodermal lineage, for example hepatic cells and/or pancreatic cells. Amniotic epithelial cells also have the ability to differentiate into cells of mesodermal lineage, for example myocytes, cardiomyocytes, osteocytes, chondrocytes and/or adipocytes. Amniotic epithelial cells may also differentiate into other cell types. The cell types into which AECs differentiate may be dependent on various factors, including culture conditions.

Amniotic epithelial cells express stem cell markers, including Oct-4. Amniotic epithelial cells may also express a variety of other markers, although the levels and pattern of marker expression may vary depending on how the cells are treated and whether or not the cells are expanded in culture. A summary of the markers typically expressed by undifferentiated human amniotic epithelial cells is shown in Table 2:

Table 2 - marker expression by undifferentiated hAECs (adapted from anuelpillai et al, Placenta 32 (2011 ), S320-325).

Stem cells obtained by methods of the invention can be identified using standard assays. In particular, testing for characteristic stem cell markers can be carried out using methods described below under "Assays for testing cells", or by any method known in the art. Preferred assays include flow cytometry and colony forming unit (CFU) assays. In preferred

embodiments, cells of the invention express the stem cell marker Oct-4. Stem cells obtained by the methods of the invention can be identified by their expression of Oct- . Stem cells of the invention may also express other cell markers, including cell surface markers. Particular markers may include those listed in Table 1 and/or Table 2. Cells of the invention may be cultured. Cells may be incubated in a suitable cell culture medium, typically on a solid surface under conditions which allow cells to adhere to the solid surface and proliferate, as described above under "Cell culture".

In preferred embodiments, cells of the invention meet good manufacturing practice (GMP) quality or standards. This is especially important where cells are to be used in a clinical setting or therapeutically. Requirements for cell therapy products are outlined in several directives and guidelines issued and periodically updated by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA), which are discussed more fully above under

"Methods". In preferred embodiments, cells of the invention are not substantially manipulated, wherein "substantially manipulated" has the sense outlined above under "Methods". In preferred embodiments, cells of the invention are minimally manipulated, wherein "minimally manipulated" has the sense outlined above under "Methods".

Total nucleated cell (TNC) count

Cells obtained by methods of the invention can be counted or quantified. Methods for counting or quantifying the obtained cells include manual methods, for example using a haemocytometer or Neubauer Chamber, or automatic methods, for example using an electronic cell counter. Electronic cell counting may be carried out, for example, using a Coulter Counter (Beckman Coulter) or a CASY^® Cell Counter and Analyser System (Roche Diagnostics GmbH).

Colony forming units (CFU)

Colony formation assays (also referred to as Colony Forming Unit (CFU) assays or Colony Forming Cell (CFC) assays) may be used to provide quantitative information about the number of stem cells obtained via methods of the invention. The colony formation assay measures proliferation in a semisolid culture medium (sometimes referred to as a matrix). Colonies are usually counted after 2-4 weeks of culture in the semi-solid medium. The culture time needs to be sufficient for sizable colonies to appear (e.g. > 40 cells/colony). As well as quantitative assessment, colonies can be analysed qualitatively, for example to check for altered appearance and size of colonies. Specific formulations of different growth factors, cytokines and other nutrients within the assay medium can determine which type of progenitor colony develops within the culture system. Commercial assays and kits are available for carrying out colony formation assays, for example the StemTag™ colony formation assays (Cell Biolabs, Inc.). Assays may also include methods for quantifying the cells that do not rely on manual counting, for example solubilising and lysing cells followed by detection of a fluorescent or colorimetric dye.

Viability

Viability of the obtained cells can be tested using a variety of standard assays known in the art, for example using a commercial kit. Commercial kits are available to measure parameters such as ATP levels (e.g. CellTiter-Glo^® Luminescent Cell Viability Assay, Promega) or metabolic activity, for example via reduction of resazurin (e.g.CellTiter-Blue^® Cell Viability Assay,

Promega) or a tetrazolium-based product such as TS (e.g.CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay, Promega). Levels of apoptotic and/or necrotic cells may also be monitored, for example via assays to measure caspase activity and/or lactate dehydrogenase (LDH) release, respectively.

Assays for cell markers

Commercially available and other known monoclonal antibodies against cell-surface markers of interest (e.g. cellular receptors and transmembrane proteins) can be used to identify and characterise amniotic stem cells.

Expression of cell surface markers may be determined, for example, by means of flow cytometry and/or FACS for a specific cell surface marker using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art) to determine whether the signal for a specific cell surface marker is greater than a background signal. The background signal is defined as the signal intensity generated by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker. For a marker to be considered positive the specific signal observed is more than 20%, preferably stronger than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity. Alternative methods for analysing expression of cell surface markers of interest include visual analysis by fluorescence microscopy using fluorescently- labelled antibodies against cell-surface markers of interest.

Cell markers (including cell surface and intracellular proteins) can be analysed by various methods known to one skilled in the art to assay protein expression, including but not limited to gel electrophoresis followed by western blotting with suitable antibodies, immunoprecipitation followed by electrophoretic analysis, and/or fluorescence microscopy as described above, with cell permeabilisation for intracellular markers. Expression of Oct-4 and/or other markers of interest, for example the markers listed in Tables 1 and 2, may be assayed using one or more of the above methods or any other method known to one skilled in the art. RNA levels may also be analysed to assess marker expression, for example by quantitative reverse transcription PCR (qRT-PCR).

Microbiology/virology

It is important to ensure that the obtained cells are not contaminated with infectious agents, particularly where these cells are to be used in therapy. Infectious agents include bacteria, fungi, parasites, mycoplasma and viruses. The obtained stem cells may therefore be tested for infectious agents.

A simple sterility test can be used to assay for the presence of bacteria and fungi. A sample of the cells can be inoculated into a suitable culture medium without antibiotics and incubated at 30-37°C for bacteria, or 20-25°C for fungi, for a suitable length of time (e.g. 14 days). Specific tests for individual infectious agents include tests for microbiological and virological markers, for example immunological tests. Infectious agents may be cultured and analysed histologically. PCR-based tests may be used to detect specific infectious agent nucleic acids. PCR-based tests are likely to give the greatest sensitivity of detection. A single type of test or a combination of one or more tests may be used.

Bacterial agents of interest for testing include, but are not limited to, Treponema pallidum, Mycobacterium tuberculosis, Neisseria gonorrhoeae and Chlamydia trachomatis. The cells may also be tested for endotoxin, for example using the LAL test. Virological tests may include, but are not limited to, tests for hepatitis viruses (HAV, HBV and HCV), human immunodeficiency viruses (HIV-1 and 2), human T-cell lymphotrophic viruses (HTLV-1 and 2), cytomegalovirus (C V), Epstein Barr virus (EBV), herpes simplex virus 1 and 2, other herpesviruses, polyomaviruses such as JC virus and BK virus, parvoviruses including parvovirus B19, coronaviruses such as SARS coronavirus, West Nile virus, adenoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus, papillomaviruses etc. Additional tests may include tests for transmissible spongiform encephalopathies, including Creutzfeldt-Jakob disease (CJD).

As well as testing the obtained cells, it is desirable to test the materials and reagents used for manipulation of the cells, as well as testing the subject from whom the amniotic membrane is derived. Cells may still be obtained from subjects found to be positive for a microbiological marker, but cells obtained from these subjects may be handled separately and/or be stored in a quarantine tank. Genetic stability

Cells obtained by methods of the invention can be analysed for genetic stability. A variety of different assays may be used to test genetic stability, alone or in any combination. Some useful assays are listed below, but additional useful assays will be apparent to those skilled in the art.

Global genome structure can be analysed by molecular cytogenetic techniques such as karyotyping. Other molecular techniques such as short tandem repeat (STR) analysis, single nucleotide polymorphism (SNP) analysis and/or resequencing analysis can be used to analyse changes in gene copy number or point mutations in nuclear and mitochondrial DNA.

Comparative genomic hybridization (CGH) can also be used to detect chromosomal aberrations that result in DNA copy number changes. Preferably, more sensitive microarray-based comparative genomic hybridization (array CGH or aCGH) is used. Quantitative PCR (qPCR) can be used to test for transgene insertion copy number. Transcription analysis can also be performed, for example by reverse transcription-PCR

(RT-PCR) and DNA sequencing, or by microarray analysis of the transcriptome. It may be of particular interest to monitor the expression of genes whose products are involved in DNA repair. Differentiation capacity

Differentiation capacity can be assayed by culturing the obtained cells in different conditions and analysing the cultured cells, for example histologically or for marker expression, to assess the cell type(s) present. Other assays may also be used to analyse the cultured cells, as outlined below or using any method known in the art.

Differentiation may be tested using commercially-available differentiation buffers. Adipogenic differentiation can be tested, for example, by culturing the obtained cells in Bullekit Adipogenic Differentiation Medium (Lonza) according to the manufacturer's instructions. Osteogenic differentation can be tested, for example, by culturing the obtained cells for three weeks in hMSC Bullekit Osteogenic Differentiation Medium (Lonza) according to the manufacturer's instructions.

Other methods are also available for testing differentiation capacity. Adipogenic differentiation can be tested, for example, by culturing cells for 2-3 weeks in DMEM supplemented with 10% FBS, 0.5 mM isobutyl-methyl xanthine, 200 μΜ indomethacin, 10^"6 M dexamethasone and lOpg/ml insulin. Osteogenic differentiation can be tested by culturing cells for 3-4 weeks in DMEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 0.2 mM ascorbic acid and 10^"8 M dexamethasone. Angiogenic differentiation can be tested, for example, by culturing cells for 7 days in DMEM supplemented with 2% FBS and 50 ng/ml VEGF. Assessment of angiogenic differentiation may be carried out by assaying for capillary formation, for example by plating cells on Matrigel™ Basement Membrane Matrix (BD Biosciences) and viewing the plated cells by optical microscopy at regular intervals over 2-3 days to assess whether capillary-like structures are formed.

Chondrogenic differentiation can be tested, for example, by micropellet formation as described by Johnstone et al, Exp Cell Res 10 (1998): 265-72. Myogenic differentiation may be tested using the methods described in Muguruma et al, Exp Hematol 31 (2003): 1323-1330.

Other assays for testing differentiation will be apparent to those skilled in the art, and the assays described herein are intended only as representative examples. The examples given should not be construed as limiting the scope of the claims or defining the cell types into which the obtained cells may differentiate.

Histological analysis

The obtained cells can be analysed histologically, either directly after they are obtained from the amniotic membrane, or after standard cell culture and/or cell culture to induce differentiation as described above. For histological analysis of obtained cells after culture, cells may be cultured in a chamber slide and then fixed for histological analysis, for example using paraformaldehyde.

Pharmaceutical compositions

The invention includes amniotic stem cell obtainable by any of the methods of the invention. These cells can be provided in pharmaceutically acceptable composition, which typically includes at least one pharmaceutically acceptable carrier and/or excipient in addition to the stem cells of the invention. A thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

Compositions will generally be in aqueous form.

Compositions may include a preservative. To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCI) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc. Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20 mM range.

The pH of a composition will generally be between 5.0 and 8.1 , and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.

The composition is preferably sterile. The composition is preferably gluten free. The

composition is preferably non-pyrogenic.

Typically, a pharmaceutical composition will be administered as a dose comprising a suitable number of cells. Suitable cell numbers per dose are, for example, between about 1 million cells and about 100 million cells per dose.

Uses in therapy and methods of treatment

The invention provides an amniotic stem cell obtainable by or obtained by a method of the invention, for use in therapy. The invention also provides a method of treatment comprising an amniotic stem cell obtainable by or obtained by any of the methods of the invention. Cells of the invention for use in therapy may be administered as part of a composition of the invention

(described above). Cells of the invention or obtained using methods of the invention may be particularly useful in therapy due to the minimal manipulation used in their preparation.

Cells derived from the amniotic membrane may have immunomodulatory properties, for example due to low or limited expression of MHC Class II and co-stimulatory molecules.

Various studies show that amniotic membrane-derived cells fail to induce allogeneic or xenogeneic immune responses in mixed lymphocyte reactions, strongly suppress lymphocyte proliferation induced by mitogens or alloantigens, and can even block maturation of monocytes into dendritic cells (Parolini et al, Stem Cells Dev 19 (2010): 143-154). Cells of the invention thus have potentially reduced risk of rejection in allogeneic or xenogeneic transplantation, a property which is advantageous in all therapeutic applications.

Cells of the invention may be used in regenerative medicine to repair tissue damage. Tissue damage may be physical damage to tissues such as skin, mucosa, bone, muscle, cartilage etc.,^' but is not limited to this type of damage and can be any kind of tissue damage repairable by stem cells of the invention. Cells of the invention with immunomodulatory properties may be particularly useful in the treatment of inflammatory disorders, and/or in the treatment of disorders where there is tissue damage with or caused by an inflammatory component. Cells of the invention may therefore be used in transplantation and tissue repair and/or regeneration after tissue damage, for example for ulcers, burns, and in ocular damage and disorders. Cells of the invention may also be used in the treatment of inflammatory disorders of the intestinal tract, including but not limited to ulcerative colitis, Crohn's disease and

inflammatory bowel disease. Cells of the invention may also be used in the treatment of tissue damage and inflammatory disorders in other tissues, for example in the treatment of pulmonary inflammatory disorders and pulmonary fibrosis; liver inflammatory disorders and hepatic fibrosis; cardiac disorders such as cardiac ischaemia and myocardial infarct; disorders involving damage to cartilage and bone, for example osteoarthritis etc.

Cells of the invention may also be used in therapy for neurological disorders, including but not limited to Parkinson's disease, stroke and spinal cord injury (SCI). Amniotic membrane-derived stem cells may be able to differentiate into angiogenic cell types, for example endothelial cells (Alviano et al, BMC Developmental Biology 7 (2007): 11-25). Cells of the invention may therefore be used in treatment of disorders where it is desirable to promote angiogenesis, for example to restore tissue damaged by various ischaemic vascular disorders. Disorders of interest include, but are not limited to, occlusive vascular diseases such as myocardial infarction, peripheral occlusive vascular disease, ischaemia (including critical limb ischaemia), stroke and other adenopathies.

Amniotic membrane-derived stem cells may be able to differentiate into haematopoietic cell types, and may therefore be used to treat blood disorders such as leukaemias, lymphomas and haemoglobinopathies, including but not limited to Sickle Cell Disease and thalassaemias.

Cells and compositions of the invention can be administered directly to the diseased site, or systemically. Various modes of administration can be used, including by not limited to intravenous administration, intramuscular administration, transdermal administration and subcutaneous administration. Optionally, the cells and compositions are administered by injection.

Cells of the invention for use in therapy may be used as an autologous treatment or as an allogeneic treatment. The term "autologous" as used herein means that the cells are used in the same patient from whom the amniotic membrane was obtained. Since amniotic membrane stem cells are of foetal origin, autologous use of the cells means use in the offspring, rather than in the mother. The term "allogeneic" as used herein, means that the cells are from a different patient than the patient from whom the amniotic membrane was obtained. The donor should usually be genetically similar to the recipient. The donor is often a sibling, but could be unrelated. However, the immunomodulatory properties of some amniotic membrane stem cells may mean that a lower degree of genetic similarity is needed between donors and recipients, compared with the degree of genetic similarity usually required for transplantation.

The invention is further described with reference to the following non-limiting examples: Examples Example 1 - obtaining human amniotic stem cells

A human placenta was placed on a sterile tray in a microbiological safety cabinet and the amniotic membrane removed from the placenta with a scalpel and forceps, using aseptic technique. The placenta was discarded as biohazardous waste. The amniotic membrane was laid out on the sterile tray. Cells were gently scraped off the surface of the amniotic membrane using a cell scraper, and directed to a corner of the sterile tray. This step was repeated on both sides of the membrane, scraping the cells into the same comer. Duibecco's modified Eagle's medium without any added FBS (DMEM, 10 ml) was added to the scraped cells and the cell suspension was transferred into a 50 ml conical tube using a 10 ml pipette.

The amniotic membrane and tray were rinsed with 25 ml DMEM to recover residual scraped cells, and the rinsate added to the population of cells above using a 25 ml pipette.

The cell suspension was evenly distributed between two centrifuge tubes. Each tube was inverted 5 times to mix the cells. The tubes containing the cell suspension were subjected to centrifugation at 400 g for 10 min.

The supernatant was removed and discarded using a 10 ml pipette, without touching or otherwise disturbing the cell pellet. Each cell pellet was resuspended in 5 ml DMEM, and the resuspended cells pooled in a new tube. Each old tube was rinsed with 5 ml DMEM to recover residual cells, and the rinsate transferred to the new tube with the resuspended cells. The rinsing step was repeated two more times for each old tube, such that the cells were resuspended in a final volume of 40 ml. A further 6 ml DMEM was added to the resuspended cells and the tube was inverted four times to mix the cells thoroughly.

A sample of 16 ml of the resuspended cells was removed for testing microbiology, viability, total nucleated cell (TNC) count and colony forming units (CFU).

The remaining cell suspension (approximately 30 ml) was injected into a freezing bag using a 50 ml syringe and a 16 gauge needle.

Origen 5% DMSO (5 ml) was added to the freezing bag using a syringe pump.

The cells in the freezing bag were frozen using a controlled rate freezer for cryopreservation.

Example 2 - testing the obtained cells

A sample of the obtained cells is tested for total nucleated cell (TNC) count, colony forming units (CFU), viability, cell markers, microbiology and virology, genetic stability and differentiation capacity.

Claims

1. A method for obtaining amniotic stem cells by physical separation of the cells from an amniotic membrane.

2. The method according to claim 1 , wherein the separation comprises agitation of the amniotic membrane.

3. The method according to claim 1 or claim 2, wherein the separation comprises scraping of the amniotic membrane.

4. The method according to any preceding claim, wherein the separation does not comprise enzymatic treatment of the amniotic membrane, such as protease treatment.

5. The method according to any preceding claim, wherein the obtained stem cell preparation is substantially free of exogenous protease activity, such as trypsin, collagenase and/or dispase activity.

6. The method according to any preceding claim, wherein the obtained stem cell preparation is substantially free of papain, collagenase, neutral protease and/or DNase.

7. The method according to any preceding claim, wherein the obtained stem cells conform to good manufacturing practice.

8. The method according to any preceding claim, comprising the following steps:

a) scraping one side or both sides of the amniotic membrane;

d) resuspending the cell pellet(s) in a buffer.

9. The method according to claim 8, comprising the additional step of rinsing the scraped amniotic membrane after step (a) and pelleting cells from the rinsate.

10. The method according to any preceding claim, wherein a sample of the obtained stem cells is removed for testing.

11. The method according to any preceding claim, further comprising cryopreservation of the obtained stem cells.

12. The method according to any preceding claim, comprising the following steps:

a) scraping one side or both sides of the amniotic membrane;

b) transferring the cells obtained from (a) to one or more sterile containers; c) rinsing the amniotic membrane to obtain cells resting on the surface of the membrane and transferring the rinsate to one or more sterile containers of (b); d) pelleting the cells, optionally by centrifugation;

e) discarding the supernatant;

f) resuspending the cell pellet(s) in a buffer;

13. The method according to any preceding claim, wherein the obtained stem cells express one or more stem cell markers.

14. The method according to any preceding claim, wherein the obtained stem cells express Oct-4.

15. The method according to any preceding claim, wherein the amniotic membrane is human.

16. A kit for obtaining amniotic stem cells by physical separation of the cells from an amniotic membrane, comprising a tool for physically separating the cells from the amniotic membrane, a sterile container for the separated cells, and instructions for carrying out the method according to any preceding claim.

17. The kit of claim 16, wherein the tool is a cell scraper.

18. The kit of claim 16 or claim 17, further comprising a sterile freezing bag and a means for transferring the obtained cells into the sterile freezing bag.

19. An amniotic stem cell obtainable by the method of any of claims 1 to 15.

20. A pharmaceutical composition comprising an amniotic stem cell according to claim 19 and a pharmaceutically acceptable carrier or excipient.

21. An amniotic stem cell according to claim 19 or a pharmaceutical composition according to claim 20 for use in therapy.