WO2009030944A1

WO2009030944A1 - Cell purification

Info

Publication number: WO2009030944A1
Application number: PCT/GB2008/050764
Authority: WO
Inventors: Peter Dunnill; Chris Mason
Original assignee: Peter Dunnill; Chris Mason
Priority date: 2007-09-05
Filing date: 2008-09-02
Publication date: 2009-03-12
Also published as: GB0717242D0

Abstract

Methods for the purification of a particular mammalian cell type from a population including at least two cell types (for example, mouse embryonic stem cells, and mouse embryonic fibroblasts) are described. The methods make use of hydrophobic interaction chromatography to selectively retard the movement of one cell type within an expanded bed. The methods may also conveniently be used for the separation of stem cells from feeder cells for use in cellular therapies.

Description

Cell purification

FIELD OF THE INVENTION

The present invention relates to a method of purifying mammalian cell populations, and in particular to a method of separating differentiated mammalian cells from stem cells and mixtures of differentiated cells from one another.

BACKGROUND TO THE INVENTION Stem cells are progenitor cells which have the ability to self-renew, and to differentiate into mature tissues. Pluripotent stem cells can differentiate into any tissue, while adult (or somatic) stem cells are more restricted and generally may differentiate into one or a few types of tissue. Examples of adult stem cells include haematopoietic stem cells, epidermal stem cells, and neural stem cells.

Cells derived from pluripotent stem cells, including those derived from embryonic and induced pluripotent stem (iPS) cells, have great therapeutic potential. However, as well as expanding the original stem cell population to the numbers of cells needed for treatment there will also be a need to ensure that no original pluripotent cells remain which might form teratomas. There may also be a need to purify differentiated cells from adult stem cells, although in this case the risk of teratomas is absent. There is also a need to be able to separate mixtures of differentiated cells.

Purification can be achieved by antibody or other ligand binding but this is costly and may cause damage to the cells, or undesirable modifications. Certain cell types (for example, lymphocytes) will attach to substrates, allowing them to be separated from non-attachment cells; however, this is only of use under restricted conditions and with a limited range of cell types. Alternatively genetic methods may be used, such as the insertion of marker genes into the cells; however, this adds greatly to the regulatory challenges, and there are risks associated with the use of transformed cell lines on patients.

A further option is physical separation of cells on the basis of size and density; for example density gradient separation, but this is poor in its separation resolution, and requires that the cell types to be separated do indeed differ sufficiently in density.

None of these purification routes are satisfactory, and there is a need for alternative methods.

Chromatographic separation is generally used in separation of molecules; for example, amino acids and other small molecule chemicals, and macromolecules such as proteins. Affinity chromatography is sometimes used for this purpose; however, this requires the use of particular ligands which will specifically bind the molecules to be separated. It is not possible to apply this technique to the separation of differentiated cells from undifferentiated cells in the absence of knowledge as to specific ligand binding markers on the desired cell type. Further, the affinity binding and elution process may itself cause damage or undesirable modification to the cells.

Preferred chromatographic methods also include packed bed chromatography, on the basis of higher resolution. To avoid clogging of the bed the particles of the chromatographic medium need to be large. This may reduce the resolution of separation.

Some chromatographic separation of cells has been reported in the literature; however, this is limited to particular cell types under restricted conditions. For example, gel filtration has been used to separate red blood cells with different deformabilities; while separation of bacteria from yeast cells has also been reported. However, such separation techniques only apply to cells of very different properties - such as size, or deformability - and resolution was limited in the case of gel filtration.

There is a need to develop a separation method which will separate cells having generally similar properties - for example, mammalian stem cells and differentiated mammalian cells. The present invention identifies such a method which makes use of non-affinity chromatographic separation, and in particularly preferred embodiments uses the hydrophobic interaction chromatography mechanism. This allows for separation of cells on the basis of differential cell surface hydrophobic interactions, and does not involve strong binding of cells as in affinity methods or to ligands with the associated potential for cell damage. Instead it rests on small differences in retention of different cells amplified by many repeats of the weak binding and release through a column of the chromatographic matrix or expanded bed. Advantages of such a method include its potential application to cell types without the need to identify specific ligands which bind the cells, or to characterize the differentiated cells in detail. Further, such a method would be generic, inexpensive and use already validated separation media.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method of purifying mammalian cells, the method comprising separating a cell population comprising a mixture of mammalian cells by means of non-affinity chromatography which includes selective retardation of the movement of cells within a chromatographic matrix.

Thus, cells of a particular type - for example, stem cells - will be differently retarded in their movement within the matrix compared with cells of another type - such as differentiated cells. In this way, the desired cell fraction(s) may be collected for further use. The non-affinity nature of the chromatography used means that the separation method is relatively gentle on the cells themselves, while also avoiding the need to identify or create specific markers for separation of cells. It is known that stem cells and differentiated cells naturally express different cell surface properties and markers, although not all of these markers have been identified. The present chromatography method makes use of the different physical properties of cells with and without such markers, but does not rely on knowledge of the markers themselves, nor on knowledge of binding targets of the markers. Indeed the markers themselves are probably unimportant in the present method.

Preferably the non-affinity chromatography is hydrophobic interaction chromatography. This relies on hydrophobic interactions between molecules (most commonly proteins, but not restricted to this) to be separated and a ligand (such as an alkyl chain) on the surface of the chromatography matrix. It will be appreciated that the degree of retardation will depend on the specific cell types to be separated, and the specific conditions of chromatography used; in order for the method to work it is merely necessary that there is some differential retardation of the cell types to be separated. The skilled person will be able to determine and to select appropriate conditions for the cell types to be used. It is preferred that biological binding of the cells to the matrix is avoided, and the retardation times may be selected with this in mind.

The chromatography may comprise eluting cells from the matrix with a suitable elution buffer or cell culture media. The cell preparation itself may also comprise a suitable buffer or cell culture media; this may be the same as or different from the elution solution. A example of a suitable solution is Dulbecco's Modified Eagle's Medium (DMEM); other examples may include phosphate buffered saline (PBS); preferably at pH 6 to 8; more preferably pH 6.5 - 7.7, pH 7.0 - 7.5, and most preferably pH 7.4.

Preferably the chromatography is carried out in a column format. The chromatography may use an expanded bed format (expanded bed absorption, or EBA). That is, the chromatography matrix is subjected to upward flow, and the cell preparation passed through the expanded bed. This reduces the risk of the bed becoming clogged by large particulates, such as the cells themselves.

The chromatography matrix preferably comprises a matrix of a polymer which may be organic or inorganic; and/or which may be natural or synthetic. The matrix preferably has one or more hydrophobic ligands thereon. The matrix may, for example, comprise cross-linked agarose, a homo- or co-polymer, or the like; for example, Sepharose ®. The ligand may comprise an alkyl or aryl group, and is preferably a Cl - C8 alkyl chain, optionally with an aryl group as a side chain. The aryl group may be phenyl, or may be naphthyl. The ligand may be charged. The alkyl may be primary, secondary, or tertiary alkyl.

The mammalian cells may be rodent cells, for example mouse cells. The mammalian cells may be primate cells, preferably human cells.

The cell preparation may comprise stem cells and non stem cells. Preferably the non stem cells are derived from stem cells of the same type as the stem cells in the preparation. In certain embodiments of the invention, the method may further comprise the step of providing a preparation comprising stem cells, and allowing at least some of the stem cells to differentiate into non stem cells. The stem cells are preferably pluripotent stem cells, and may be embryonic or induced pluripotent stem cells; or may be adult stem cells, for example, haematopoietic stem cells or neural stem cells. Alternatively, or in addition, the non stem cells may be unrelated to the stem cells; for example, the non stem cells may be feeder cells such as embryonic fibroblast cells, on which the stem cells may be cultured; in certain embodiments, the stem cells may be human, and the feeder layer murine. The cell preparation may comprise induced pluripotent stem cells (iPS cells); that is, cells demonstrating ES-like potency derived from differentiated cells, for example, mouse fibroblasts. The method may be used to separate iPS cells from differentiated cells.

The method may also be used to separate partially differentiated cells (for example, progenitor cells, or lineage-committed cells) from undifferentiated stem cells or from differentiated cells.

The method may comprise collecting one or more fractions of the cell preparation that have passed through the chromatography matrix.

A further aspect of the invention provides a method for separating differentiated mammalian cells from a preparation comprising mammalian stem cells and differentiated mammalian cells, the method comprising passing the preparation through a hydrophobic interaction chromatography matrix such that either the stem cells or the differentiated cells are retarded in their passage compared with the remaining cells.

The method may also comprise the step of providing a preparation comprising stem cells, and allowing at least some of the stem cells to differentiate, to provide the preparation comprising stem cells and differentiated cells.

The method may further comprise the step of eluting cells from the matrix using a suitable buffer.

One or more fractions of the cell preparation may be collected.

The present invention further provides a method for obtaining a purified preparation of differentiated mammalian cells, the method comprising: providing a population comprising mammalian stem cells; allowing at least some of the stem cells to differentiate, to obtain a preparation of stem cells and differentiated cells; passing the preparation through a hydrophobic interaction chromatography matrix such that either the stem cells or the differentiated cells are retarded in their passage compared with the remaining cells; and collecting a fraction comprising differentiated cells.

Also provided is a method for separating differentiated mammalian cells from mammalian stem cells, the method comprising separating a cell population comprising a mixture of differentiated mammalian cells and mammalian stem cells by means of non-affinity chromatography which includes selective retardation of the movement of either of the stem cells or differentiated cells within a chromatographic matrix.

Also provided is a method for separating cell types from a mixture of differentiated mammalian cells, the method comprising separating a cell population comprising a mixture of types of differentiated mammalian cells by means of non-affinity chromatography which includes selective retardation of the movement of the types of differentiated cells within a chromatographic matrix.

The invention also provides a method for separating mammalian stem cells from non stem cells, the method comprising passing a preparation comprising mammalian stem cells and non stem cells through a hydrophobic interaction chromatography matrix such that either the stem cells or the non stem cells are retarded in their passage compared with the remaining cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the comparative binding of undifferentiated mouse embryonic stem cells (mESC) and a differentiated population of mouse embryonic fibroblast cells (mEF) to butyl sepharose chromatography beads. Figure 2 shows the results obtained using phenyl sepharose beads. Figure 3 shows the results obtained using octyl sepharose beads.

DETAILED DESCRIPTION OF THE INVENTION

Human cells show potential for therapy of previously untreatable conditions or those where the outcome is inadequate. In particular, human stem cells are of interest because they can be transformed into a range of specialised cells. This potential is most marked with human embryonic or induced pluripotent stem cells and, whereas adult stem cells have a limited capacity to reproduce, embryonic cells can be expanded indefinitely. Because the cells used for therapy will be living it is critical that they do not change into cancerous cells or other aberrant forms. This means that in addition to steps which multiply the number of cells there is a need for methods which selectively purify particular cells or alternatively remove certain unwanted ones. This can be achieved by genetic means but that adds to the regulatory challenges. It may also be achieved using binding to antibodies which are themselves immobilised, but this is costly and separating the cells subsequently from the antibody may cause damage or undesirable change. It may be possible to produce a single type of cell from the embryo but this remains to be established. Finally a physical separation may be used but methods such as density gradient centrifugal sedimentation produce limited resolution when applied to cells. It would therefore be valuable to have a general method of higher resolution separation which did not entail affinity binding of the kind involved with antibodies or other special agents.

The method described herein is based on the automatic cascade operation of chromatography or its related expanded bed format. Packed beds used in chromatography give greater resolution of separation than expanded beds but can be clogged by cells. To achieve the necessary resolution requires generally a chromatographic or related method such as hydrophobic or ion- exchange separation. Gel filtration generally lacks resolution except where size differences are relatively large but has been applied to red blood cells with different deformabilities (Rohner et al, 1990). There is a report that cells have been separated by batch sorption-desorption on hydrophobic media (Halperin et al, 1984) and ion-exchange binding has been noted as an interference in expanded bed adsorption of proteins (Fenser et al, 1999). However, the aim with the latter paper was not cell separation and with Halperin et al, 1984 the main interest was to probe cell surface properties. The test cells used were erythrocytes, which are non-attachment, and lymphocytes which are attachment cells under appropriate conditions. Hjerten (1981) noted the possibility of separating particles including cells by hydrophobic interaction chromatography commenting that with large enough agarose beads the particles can pass through the bed; however, the only data given relates to yeast cells, which as fungal cells are very different from mammalian cells, and can withstand more disruptive treatment. The proposed method would not be suitable for mammalian cells.

Two papers use chromatographic methods to separate bacteria (Stjernstrom et al, 1977 and Smyth et al 1978). The paper of Smyth et al exploits differences between E. coli strains where there is a single surface antigen difference. These are principally concerned with analysis of cell surfaces and use high ionic strength solutions which would damage human and mammalian cells. Halperin et al, 1976 showed that differences could be seen in a batch procedure with erythrocytes from two inbred strains of mice. An expanded bed ion-exchanger has been used to separate E. coli and S. cerevisiae (Ujam et al, 2000). Affinity chromatography has been widely applied to biological cells (see Sharma and Mahendroo, 1980). However, it is usually system specific, expensive and because of the very strong binding the detachment process can lead to damage to the cell or undesirable change.

The present invention is principally concerned with separating closely related human cells, although it may be applied to other mammalian cells, as well as to separation of stem cells from feeder cells. A particular concern in the new field of regenerative medicine is the possibility of residual human pluripotent stem cells remaining with cells differentiated into the specialized cells needed for therapy. These pluripotent cells have a tendency to form potentially harmful teratomas. The difference between pluripotent and specialised cells is small and hence the multi step cascade separation of chromatography or expanded bed separation is likely to be needed. The issue of risk when human cell "contaminants" are present is addressed in a recent paper (Hentze et al, 2006) and will be central for clinical progress in the field.

EXAMPLES

The principle of the method is demonstrated in two studies both examining the separation of mouse embryonic stem cells (mESCs) which can be grown without a feeder layer from mouse embryonic fibroblast cells (mEFs) of the type that are commonly used as feeder layers for embryonic stem cells that require such a layer. These studies are used to establish whether undifferentiated, and potentially tumorigenic, embryonic stem cells can be separated from differentiated cells prior to their application in cellular therapies.

In the first study batch adsorption was used to characterise the system and establish the likely conditions and materials for chromatography which is then undertaken in the second study. The hydrophobic interaction chromatography (HIC) media used were commercial GE Healthcare products available with variable alkyl chains. The study compared the binding of mESC and mEF to butyl, phenyl and octyl sepharose beads in batch processes.

In low attachment 96 well plates samples of 0.1 ml of cells at 10⁴, 10⁵, 10⁶ and 10⁷ cells/ml were added to 0.1ml of HIC media from a stock of 10⁵ beads/ml. For all of the experiments the both the cells and the HIC media were buffered in Dulbecco's Modified Eagle Medium (DMEM). The cell-HIC media mixture was incubated for 15 minutes at 37⁰C. After this period the number of cells attached per bead were analysed by light microscopy. All batch adsorption experiments were completed in triplicate. The isotherms in figures 1, 2 and 3 were generated by plotting the cells bound per bead (Q) against the final concentration of cells. For butyl, phenyl and octyl sepharose the binding of both cell types increased rapidly at low concentrations and plateaued beyond 5 x 10⁶ cells/ml. The differentiated population of cells, mEF, consistently bound more readily to all three HIC media than the undifferentiated cells, the mESC. In addition the binding of both cell types increased with increasing alkyl chain length with octyl sepharose exhibiting the strongest affinity.

The adsorption isotherms described are the standard means by which chromatography performance is defined and together with the known art they provide the basis of any chromatographic or expanded bed separation.

PROPHETIC EXAMPLE

The batch adsorption provides information on the binding of cells with the range of HIC adsorbents. Based on this either packed bed or expanded bed chromatography is undertaken with the octyl-sepharose media which exhibited the largest differential binding between the two cell types. The volumes of adsorbent and the flow rates are adjusted to maximise the separation consistent with avoiding excessive dilution. mESC have the least affinity to the HIC media and will elute first, followed by a second peak of differentiated mEF cells. Once applied to human cells the potentially tumorigenic human pluripotent cells can be eluted before collecting the differentiated population for subsequent processing or transplantation as a cellular therapy. Other variations to the process which can affect the binding include temperature, pH, the presence of hydrophobic proteins which can competitively bind to the beads and ionic strength.

It will be appreciated that similar experiments may be undertaken for other mixtures of cells, including stem cells and cells differentiated from such stem cells. This will also be effective for human pluripotent stem cells, as well as for somatic stem cells. The determination of appropriate conditions to obtain a desired separation of cells, and hence a desired retardation of one cell type compared with another, is within the ability of the skilled person.

The skilled person will be aware of other variations which may be made to the described procedure.

References

Feuser, 1, Walter, J., KuIa, M. R., Thommes, J. 1999. Cell/adsorbent interactions in expanded bed adsorption of proteins. Bioseparation. 8, 99-109.

Halperin, G., Shaltiel, S. 1976. Homologous series of alkyl agaroses discriminate between erythrocytes from different sources. Biochem. Biophys. Res. Commun. 72, 1497-503.

Halperin, G., Tauber-Finkelstein, M., Shaltiel, S. 1984. Hydrophobic chromatography of cells: Adsorption and resolution on homologous series of alkylagaroses. J. Chromatog. 317, 103-118.

Hentze, H., Graichen, R., Colman, A. 2006. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotech. 25, 24-32.

Hjerten, S. 1981. Hydrophobic interaction chromatography of proteins, nucleic acids, viruses, and cells on noncharged amphiphilic gels. Methods Biochem. Anal. 27, 89-108.

Rohner, F., Reinhart, W.H., Haeberli, A., Straub, P.W. 1990. Gel filtration: A new method to analyze and separate red blood cells with different deformabilities. J Lab Clin Med. 116, 393-9.

Sharma, S.K., Mahendroo, P. D. 1980. Affinity chromatography of cells and cell membranes. J. Chromatog. 184, 471-499.

Smyth, CJ., Jonsson, PJ., Olsson, E., Soderlind, U., Rosengren, J., Hjerten, S., Wadstrom, T. 1978. Differences in hydrophobic surface characteristics of porcine enteropathogenic Escherichia coli with or without K88 antigen as revealed by hydrophobic interaction chromatography. Infect Immun. 22, 462- 472. Stjernstrom, L, Magnusson, K.E., Stendahl, 0., Tagesson, C. 1977. Liability to hydrophobic and charge interaction of smooth Salmonella typhimurium 395 MS sensitized with anti-MS immunoglobulin G and complement. Infect Immun. 18, 261-265.

Ujam, B., Clemmittr, R.H., Chase, H.A. 2000. Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of E. coli and S. cerevisiae. Bioprocess Eng. 23, 245-250.

Claims

1. A method of purifying mammalian cells, the method comprising separating a cell population comprising a mixture of at least two types of mammalian cells by means of non-affinity chromatography which includes selective retardation of the movement of cells within a chromatographic matrix or expanded bed.

2. The method of claim 1, wherein the non-affinity chromatography is hydrophobic interaction chromatography in a packed or expanded bed.

3. The method of claim 1 or 2, wherein the cells in the preparation to be separated are retarded for sufficiently short a time to avoid biological binding of the cells to the matrix.

4. The method of any preceding claim further comprising eluting cells from the matrix with a suitable elution buffer.

5. The method of any preceding claim, wherein the chromatography uses an expanded bed format.

6. The method of any preceding claim wherein the chromatography matrix comprises a matrix of natural or synthetic polymer having one or more hydrophobic ligands thereon.

7. The method of any preceding claim wherein the chromatography matrix comprises a matrix of organic or inorganic polymer having one or more hydrophobic ligands thereon.

8. The method of any preceding claim wherein the mammalian cells are rodent cells.

9. The method of any of claims 1 to 7 wherein the mammalian cells are primate cells.

10. The method of claim 9 wherein the cells are human cells.

11. The method of any preceding claim wherein the cell preparation comprises stem cells and non stem cells.

12. The method of claim 11 wherein the non stem cells are derived from stem cells of the same type as the stem cells in the preparation.

13. The method of claim 11 wherein the non stem cells are unrelated to the stem cells.

14. The method of any preceding claim wherein the preparation comprises induced pluripotent stem cells.

15. The method of any preceding claim comprising collecting one or more fractions of the cell preparation that have passed through the chromatography matrix.

16. A method for separating differentiated mammalian cells from a preparation comprising mammalian stem cells and differentiated mammalian cells, the method comprising passing the preparation through a hydrophobic interaction chromatography matrix such that either the stem cells or the differentiated cells are retarded in their passage compared with the remaining cells.

17. A method for obtaining a purified preparation of differentiated mammalian cells, the method comprising: providing a population comprising mammalian stem cells; allowing at least some of the stem cells to differentiate, to obtain a preparation of stem cells and differentiated cells; passing the preparation through a hydrophobic interaction chromatography matrix such that either the stem cells or the differentiated cells are retarded in their passage compared with the remaining cells; and collecting a fraction comprising differentiated cells.

18. A method for separating cell types from a mixture of differentiated mammalian cells, the method comprising separating a cell population comprising a mixture of types of differentiated mammalian cells by means of non-affinity chromatography which includes selective retardation of the movement of the types of differentiated cells within a chromatographic matrix.