- Email: [email protected]
Immunomagnetic cell sorting—pushing the limits
Immunotechnology 4 (1998) 89 – 96
Review article
Immunomagnetic cell sorting—pushing the limits Andreas Thiel *, Alexander Scheffold, Andreas Radbruch Deutsches Rheuma-Forschungszentrum Berlin, Hanno6ersche Straße 27, 10115 Berlin, Germany Received 5 February 1998; accepted 13 February 1998
Abstract Efficient cell separation is a prerequisite to the functional analysis of specialised cell types within complex biological systems. High gradient magnetic cell sorting (MACS) has become increasingly popular as the method of choice for cell separation for various applications. It combines high sensitivity and high purity as well as increased recovery and viability of isolated cell populations compared to other methods. Magnetic cell sorting is particularly useful for isolation of rare cells from heterogeneous cell populations. The recent development of MACS-MultiSort eliminated the last drawback of magnetic cell sorting, namely the restriction to a single parameter. The option to sort for multiple parameters has now been added to the advantages of this method. Additionally, technologies have been developed for isolating live cells according to secreted products or for analysing and separating cells expressing antigens at very low density. These new techniques add new parameters for cytometric analysis and together with efficient magnetic separation they provide new perspectives for research, as well as for the diagnosis and therapy of many diseases. Here we discuss some of these advanced methods, their potential and their limitations. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cell separation; High gradient magnetic cell sorting; Rare cell isolation
1. Conventional cell separation methods Apart from physical (density gradient centrifugation) and biochemical (adherence, erythrocyte lysis) [1] methods for cell separation, techniques * Corresponding author. Tel.: +49 30 28518966/8964; fax: + 49 30 28518910; e-mail: [email protected]
based on immunological recognition for specific labeling and separation have gained in significance during the last few years. Cells specifically labeled with fluorochrome coupled antibodies can be separated with a fluorescence-activated cell sorter (FACS). Although the FACS-technology provides impressive results regarding purity of separated cells, it is limited by the time required
1380-2933/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S1380-2933(98)00010-4
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for separation which can be several hours per sample. Being a serial sorting device (cells are analysed and sorted one by one), the capacity of FACS is limited by the speed of analysis and sorting which is about 5000 cells per s, i.e. 108 cells in 6 h, or with less purity, performing ‘high speed’ separations 5×108 cells in 6 h. As a consequence, several ‘parallel’ sorting technologies had been developed, which can process even very high cell numbers through parallel separation of labelled cells. This type of cell isolation is based on the separation with macroscopic immunospecific surfaces [2] or macroscopic magnetic particels [3,4]. However, large surface areas are involved in the process of cell isolation and these tend to bind cells unspecifically. Therefore it is difficult to achieve high enrichment rates, especially when performing rare cell isolation.
2. High gradient magnetic cell separation (MACS) The introduction of colloidal magnetic particles of less than 100 nm in diameter conjugated to specific ligands or antibodies, allowed to combine the advantages of the methods described before [5,6]. The magnetic label does not interfere with FACS analysis nor does it alter functional properties of cells. The small size of the particles permits quantitative and highly specific labeling [1,6,7]. Because labeled cells are processed in parallel,
Fig. 1. The principle of high gradient magnetic cell sorting (MACS). A mixture of magnetically labeled and non-labeled cells is applied on a separation column (A). Magnetically labeled cells are retained in the magnetic field of the separation column; non-labeled cells are isolated as negative (depleted) fraction (B). After removal of the column from the magnetic field, labeled cells are eluted (C).
high cell numbers can be processed within a short time. The recent development of multiparameter high gradient magnetic cell sorting (MACS-MultiSort) eliminated an inherent drawback of magnetic cell sorting, which was the restriction to a single parameter [8]. Multiparameter high gradient magnetic cell sorting (MACS-MultiSort) adds a new and powerful alternative to conventional fluorescence-activated cell sorting (FACS) and pushes the limit of cell sorting, especially for the
Fig. 2. Isolation of CD34 + cells from normal peripheral blood. Flow cytometric analysis of peripheral blood mononuclear cells (PBMC) stained with CD34PE (A) before separation, (B) positive fraction, eluted from first separation column and (C) positive fraction after a second separation round. FL2 fluorescence is plotted versus side scatter (SSC). Dead cells and debris are excluded from the analysis.
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Fig. 3. The principle of multiparameter high gradient magnetic cell sorting (MACS-MultiSort). After a first separation (as illustrated in Fig. 1), magnetic MicroBeads are released from the cell surface by enzymatic treatment (A). Cells are magnetically labeled for a second parameter and applied on a new separation column (B). Only cells expressing the second marker are retained in the magnetic field; non-labeled cells are isolated as negative (depleted) fraction (C). After removal of the column from the magnetic field labeled cells are eluted (D).
isolation of rare cells and the processing of large cell numbers.
3. Method The magnetic label is provided by superparamagnetic beads conjugated to specific ligands or antibodies. Cells are separated on ferromagnetic matrices in a high-gradient magnetic field. Nonmagnetic cells pass this matrix, whereas magnetically tagged cells are retained and subsequently eluted after removal of the column from the magnetic field (Fig. 1). This technology allows the processing of cell numbers above 1010 in a short time.
4. Purity, viability and functional potential of isolated cells Not only can high purities be achieved, cells are also separated in a gentle way. No strong mechanical forces influence cell integrity or viability. Because all cells are sorted simultaneously, up to 1011
cells can be processed in about 30 min, giving this method a leading edge in the sorting of rare cells. The little physical stress during MACS sorting favors the recovery of viable cells, unlike FACSsorting, where the cells are submitted to considerable stress by acceleration in the nozzle. Magnetically sorted cells have been used to generate dendritic cells from CD14 + monocytes [9], to analyse cytokine secretion of T-cell subsets and the cytokine secretion patterns of CD61 expressing megakaryocyte progenitors [10], to investigate Tcell–B-cell interactions in complex in vitro culture systems [11,12], to expand human haematopoietic progenitor cells ex vivo [13] and even for cell transfer experiments [14]. A recent development is a clinical grade separation system which performs the separation of CD34 expressing human haematopoietic progenitor cells from various sources and will be used in cellular therapy of human malignant or inherited diseases [15].
5. Isolation of rare cells The potential of high gradient magnetic cell
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Fig. 4. Isolation of CD4 + CD45RO − CD45RA + T-cells from human PBMC using multiparameter magnetic cell sorting. Cells are stained with CD4 PE (A, B) or CD45RO PE and CD45RA FITC (C, D). A: PBMC before CD4 separation. B: positive fraction after MACS enrichment of CD4 + Th cells. C: after removal of CD4 specific MicroBeads cells are stained with CD45RO specific MicroBeads, PE conjugated CD45RO and FITC conjugated CD45RA FITC. D: negative fraction after MACS depletion of CD45RO + cells.
sorting is best recognised when sorting of rare cells is desired. Here, if the frequency of positive cells becomes very low ( B 0.1 – 1%), highly efficient labeling of the desired cell population paired with a short processing time is a prereq-
uisite of successful isolation. CD34 expressing haematopoietic progenitor cells in normal human peripheral blood represent only 0.1% of peripheral mononuclear cells (PBMC). However, the MACS-technology allows an efficient isola-
A. Thiel et al. / Immunotechnology 4 (1998) 89–96
93
Fig. 5. Isolation of CD34 + HLA–DR − cells from human PBMC using multiparameter magnetic cell sorting. Cells are stained with CD34 PE and HLA – DR FITC. A: MACS isolated CD34 + cells before MACS depletion of HLA – DR − CD34 + cells. B: negative fraction after depletion of HLA–DR − CD34 + cells. (The frequency of CD34 + cells among PBMC is about 0.1%; thus HLA – DR − CD34 + cells represent about 0.001% of total PBMC).
tion of this cell population (Fig. 2). Functional tests reveal excellent viability of the isolated cells [13,16]. For the isolation of CD34 + cells from various tissues and from peripheral blood of normal donors no other method is available that achieves comparable enrichment rates (up to 20000-fold) and recoveries [17 – 19]. Allergen-specific B-cells from the peripheral blood of atopic donors (1 in 10 − 4) [20] and fetal cells from the peripheral blood of pregnant woman (1 in 10 − 5) [21,22] have been isolated successfully. Recently a method has been developed for the enrichment of tumor cells in peripheral blood, decreasing the detection limit for residual cancer cells in tumor patients [30].
A new challenge for cell separation is the isolation of transfected cells according to surface marker expression. Conventional selection of transfected cells by cotransfection with drug resistance genes (Gregor Siebenkotten, personal communication) is a very time consuming procedure. Recent studies suggest that magnetic cell sorting is in fact a powerful strategy for the isolation of transfected cells. Cells are cotransfected with the vector of interest and an expression vector for surface molecules, which can be specifically labeled with magnetic microparticles. At defined timepoints after transfection, cells expressing the marker molecule can be isolated, even if the rate of transfection is low. Separated cells can directly be analysed functionally, thereby eliminating the
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influence of growth advantages of cells that are not transfected (Gregor Siebenkotten, personal communication).
Fig. 6. The principle of the ‘affinity-matrix-technology’. Secreted products are ‘caught’ on the cell surface by the affinity matrix created by ‘catch’ antibodies and subsequently detected by labeling with a second ‘detection antibody’ conjugated to MicroBeads or fluorochromes. Fig. 8. Principle of signal enhancement by magnetofluorescent liposomes. A: conventional reagents bind only 1 – 10 fluorochromes per antibody. B: liposomes conjugated to specific antibodies are filled with several thousand fluorescein molecules which drastically increases the fluorescence signal intensity of labeled cells compared to conventional antibody conjugates.
6. Multiparameter high gradient magnetic cell sorting
Fig. 7. Magnetic cell separation of murine Th splenocytes according to secreted IL-2. Cells are stained for secreted IL-2 caught by the affinity matrix. A: polyclonally activated murine spleen cells before IL-2 separation. B: positive fraction of MACS separated IL-2 producing murine splenocytes.
The recent development of multiparameter magnetic cell sorting (MACS-MultiSort) eliminated the last drawback of magnetic cell sorting, namely the restriction to a single parameter [8]. After the first step of isolation, the primary magnetic labeling is removed enzymatically. A secondary magnetic labeling can now be performed as illustrated in Fig. 3. Fig. 4 shows an example of multiparameter high gradient magnetic cell sorting. The CD45RO − CD45RA + subset of CD4 + Th cells is efficiently separated from CD45RO + CD45RA − CD4 Th cells. Multiparameter sorting allows also the isolation of extremely rare subsets of cells. For example, uncommitted subsets of human haematopoietic progenitor cells represent
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Fig. 9. Staining of IL-6 receptor: conventional staining versus magnetofluorescent liposomes. Purified CD34 + cells are stained with CD34 Cy-Chrome and A: anti IL-6R PE or B: anti IL-6R DIG + anti DIG liposomes.
extremely rare cell populations. Only three cells in 105 PBMC are characterised by CD34 expression and lack of HLA–DR expression. The enrichment of such cells from an initial frequency of 0.003 – 96% (Fig. 5) stands for an 800000-fold enrichment rate.
products for a short time in vitro, and the secreted product is caught on the surface by the affinity matrix. It can then be labeled and analysed with fluorochrome or magnetically tagged antibodies. Using this technology, antibody secreting cells or activated, IL-2 (Fig. 7) or IFN-g producing T-cells have been isolated and characterised functionally [26].
7. New parameters
7.2. Magnetofluorescent liposomes Immunofluorescent analysis and sorting of live cells can be limited: either suitable surface markers are expressed only in low density or the antigen of interest is not expressed on the cell surface, e.g. secreted molecules like cytokines, only allowing analysis in fixed cells after permeabilization [23]. Recently, new technologies have been developed to overcome these limitations for analytical and preparative cytometry.
7.1. Sorting of li6e cells according to secreted products The first method for the analysis and isolation of live cells according to their secreted products was published by our group [24]. We have recently shown that this method can easily be adapted to the analysis and separation of cells secreting antibodies, cytokines or growth factors [25]. The labeling of cells according to the products they secrete can be performed by using the Biotin – Avidin-system to attach an affinity matrix onto the cell surface (Fig. 6). The cells are allowed to secrete
The detection limit of conventional immunofluorescence is in the range of several thousand molecules per cell depending on cellular autofluorescence [27]. Therefore many functionally important molecules, e.g. receptors for cytokines, hormones or growth factors were difficult to analyse, because most of them are expressed in low copy numbers. We have developed a sensitive reagent, magnetofluorescent liposomes, conjugated to specific antibodies which increase fluorescence signal intensity up to 1000-fold (Figs. 8 and 9) [28]. Thus magnetofluorescent liposomes allow clear detection of cell subsets expressing 300–400 antigens or less. In addition, they offer the possibility of sorting out labeled cells magnetically. Liposomes have been used to demonstrate for the first time the specific expression of IFN-g on the surface of IFN-g producing Th1 cells [29]. It was also possible to identify and separate functional distinct subpopulations of CD34 + hematopoietic stem cells according to the expression of receptors for IL-6 (A. Thiel, manuscript in preparation).
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8. Summary Separation of specific cell populations is a prerequisite for many analytical and functional studies in basic research as well as for diagnostic and therapeutic applications. The development of high gradient magnetic cell sorting during the last years offers a highly specific, gentle and fast way of purification of a large panel of different cell types from complex cell mixtures. Standard separations can be done within minutes using either positive or negative enrichment strategies. The magnetic particles influence neither viability or function of labeled cells nor analytical parameters of flow-cytometry. The high specificity of magnetic labeling and the short separation time result in fast processing of high cell numbers therefore allowing the purification of even rare cell populations within 1 – 2 h. In addition, the new technologies described here, e.g. magnetofluorescent liposomes for the detection of rare antigens, the ‘affinity matrix technology’ to detect secreted molecules and multiparameter magnetic cell sorting raise the number of parameters which can be used for identification and separation of specific cell populations.
Acknowledgements Many thanks to Florian Kern and Christine Raulfs for critically reading the manuscript. This work was supported by a grant from the ‘Deutsche Krebshilfe e.V.’ (W8/94/RA1).
References [1] Radbruch A, editor. Flow cytometry and cell sorting, Heidelberg: Springer, 1992. [2] Silvestri FF, Banavali SD, Hulette BC, Civin CI, Preisler HD. Int J Cell Cloning 1991;9:474–90. [3] Smeland EB, Funderud S, Kvalheim G, Gaudernack G, Rasmussen AM, Rusten L, Wang MY, Tindle RW, Blomhoff HK, Egeland T. Leukemia 1992;6:845–52. [4] Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D, Knitter G, Buckner CD, Bernstein ID. J Clin Invest 1988;81:951 – 5. [5] Molday RS, Molday LL. FEBS Lett 1984;170:232–8. [6] Miltenyi S, Mu¨ller W, Weichel W, Radbruch A. Cytometry 1990;11:231 – 8. .
[7] Radbruch A, Mechtold B, Thiel A, Miltenyi S, Pfluger E. In: Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Methods Cell Biology. San Diego: Academic Press, 1997:387 – 402. [8] Thiel A, Schmitz J, Miltenyi S, Radbruch A. Immunol Lett 1997;57:189 – 92. [9] Pickl WF, Majdic O, Kohl P, Stockl J, Riedl E, Scheinecker C, Bello-Fernandez C, Knapp W. J Immunol 1996;157:3850– 9. [10] Schmitz B, Radbruch A, Ku¨mmel T, Wickenhauser C, Korb H, Hansmann ML, Thiele J, Fischer R. Eur J Haematol 1994;52:267 – 75. [11] Blanchard D, Gaillard C, Hermann P, Banchereau J. Eur J Immunol 1994;24:330 – 5. [12] Defrance T, Vanbervliet B, Briere F, Durand I, Rousset F, Banchereau J. J Exp Med 1992;175:671 – 82. [13] Servida F, Soligo D, Caneva L, Bertolini F, de Harven E, Campiglio S, Corsini C, Deliliers GL. Stem Cells 1996;14:430 – 8. [14] Schmitz J, Thiel A, Kuhn R, Rajewsky K, Muller W, Assenmacher M, Radbruch A. J Exp Med 1994;179:1349– 53. [15] McNiece I, Briddell R, Stoney G, Kern B, Zilm K, Recktenwald D, Miltenyi S. J Hematother 1997;6:5 – 11. [16] de Wynter EA, Coutinho LH, Pei X, Marsh JC, Hows J, Luft T, Testa NG. Stem Cells (Dayt) 1995;13:524 – 32. [17] Kato K, Radbruch A. Cytometry 1993;14:384 – 92. [18] Radbruch A, Mechtold B, Thiel A, Miltenyi S, Pfluger E. In: Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Methods Cell Biology. San Diego: Academic Press, 1997:387 – 402. [19] Thiele J, Wickenhauser C, Baldus SE, Kuemmel T, Zierbes T, Drebber U, Wirtz R, Thiel A, Hansmann ML, Fischer R. Leuk Lymphoma 1995;16:483 – 91. [20] Irsch J, Hunzelmann N, Tesch H, Merk H, Maggi E, Ruffilli A, Radbruch A. Immunotechnology 1996;1:115 – 25. [21] Busch J, Huber P, Pfluger E, Miltenyi S, Holtz J, Radbruch A. Prenatal Diagn 1994;14:1129 – 40. [22] Ganshirt-Ahlert D, Borjesson-Stoll R, Burschyk M, Dohr A, Garritsen HS, Helmer E, Miny P, Velasco M, Walde C, Patterson D, et al. Am J Reprod Immunol 1993;30:194 – 201. [23] Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. J Immunol Methods 1993;166:201 – 14. [24] Manz R, Assenmacher M, Pflu¨ger E, Miltenyi S, Radbruch A. Proc Natl Acad Sci 1995;92:1915 – 21. [25] Manz RA, Thiel A, Radbruch A. Nature 1997;388:133 – 4. [26] Assemacher M, Lo¨hning M, Scheffold A, Manz A, Schmitz J, Radbruch A. Eur J Immunol 1998;28:1534 – 43. [27] Scheffold A, Miltenyi S, Radbruch A. Immunotechnology 1996;1:127 – 37. [28] Assenmacher M, Scheffold A, Schmitz J, Segura Checa J A, Miltenyi S, Radbruch A. Eur J. Immunol 1996;26:263 – 7. [29] Assenmacher M, Scheffold A, Schmitz J, Checa JS, Miltenyi S, Radruch A. Eur J Immunol 1996;26:263 – 7. [30] Martin V, Siewert C, Scharl A, Harms T, Heinze R, O8 hl S, Radruch A, Miltenyi A, Schmitz J. J Exp Hematol1998;26:252– 64.
Review article
Immunomagnetic cell sorting—pushing the limits Andreas Thiel *, Alexander Scheffold, Andreas Radbruch Deutsches Rheuma-Forschungszentrum Berlin, Hanno6ersche Straße 27, 10115 Berlin, Germany Received 5 February 1998; accepted 13 February 1998
Abstract Efficient cell separation is a prerequisite to the functional analysis of specialised cell types within complex biological systems. High gradient magnetic cell sorting (MACS) has become increasingly popular as the method of choice for cell separation for various applications. It combines high sensitivity and high purity as well as increased recovery and viability of isolated cell populations compared to other methods. Magnetic cell sorting is particularly useful for isolation of rare cells from heterogeneous cell populations. The recent development of MACS-MultiSort eliminated the last drawback of magnetic cell sorting, namely the restriction to a single parameter. The option to sort for multiple parameters has now been added to the advantages of this method. Additionally, technologies have been developed for isolating live cells according to secreted products or for analysing and separating cells expressing antigens at very low density. These new techniques add new parameters for cytometric analysis and together with efficient magnetic separation they provide new perspectives for research, as well as for the diagnosis and therapy of many diseases. Here we discuss some of these advanced methods, their potential and their limitations. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cell separation; High gradient magnetic cell sorting; Rare cell isolation
1. Conventional cell separation methods Apart from physical (density gradient centrifugation) and biochemical (adherence, erythrocyte lysis) [1] methods for cell separation, techniques * Corresponding author. Tel.: +49 30 28518966/8964; fax: + 49 30 28518910; e-mail: [email protected]
based on immunological recognition for specific labeling and separation have gained in significance during the last few years. Cells specifically labeled with fluorochrome coupled antibodies can be separated with a fluorescence-activated cell sorter (FACS). Although the FACS-technology provides impressive results regarding purity of separated cells, it is limited by the time required
1380-2933/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S1380-2933(98)00010-4
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for separation which can be several hours per sample. Being a serial sorting device (cells are analysed and sorted one by one), the capacity of FACS is limited by the speed of analysis and sorting which is about 5000 cells per s, i.e. 108 cells in 6 h, or with less purity, performing ‘high speed’ separations 5×108 cells in 6 h. As a consequence, several ‘parallel’ sorting technologies had been developed, which can process even very high cell numbers through parallel separation of labelled cells. This type of cell isolation is based on the separation with macroscopic immunospecific surfaces [2] or macroscopic magnetic particels [3,4]. However, large surface areas are involved in the process of cell isolation and these tend to bind cells unspecifically. Therefore it is difficult to achieve high enrichment rates, especially when performing rare cell isolation.
2. High gradient magnetic cell separation (MACS) The introduction of colloidal magnetic particles of less than 100 nm in diameter conjugated to specific ligands or antibodies, allowed to combine the advantages of the methods described before [5,6]. The magnetic label does not interfere with FACS analysis nor does it alter functional properties of cells. The small size of the particles permits quantitative and highly specific labeling [1,6,7]. Because labeled cells are processed in parallel,
Fig. 1. The principle of high gradient magnetic cell sorting (MACS). A mixture of magnetically labeled and non-labeled cells is applied on a separation column (A). Magnetically labeled cells are retained in the magnetic field of the separation column; non-labeled cells are isolated as negative (depleted) fraction (B). After removal of the column from the magnetic field, labeled cells are eluted (C).
high cell numbers can be processed within a short time. The recent development of multiparameter high gradient magnetic cell sorting (MACS-MultiSort) eliminated an inherent drawback of magnetic cell sorting, which was the restriction to a single parameter [8]. Multiparameter high gradient magnetic cell sorting (MACS-MultiSort) adds a new and powerful alternative to conventional fluorescence-activated cell sorting (FACS) and pushes the limit of cell sorting, especially for the
Fig. 2. Isolation of CD34 + cells from normal peripheral blood. Flow cytometric analysis of peripheral blood mononuclear cells (PBMC) stained with CD34PE (A) before separation, (B) positive fraction, eluted from first separation column and (C) positive fraction after a second separation round. FL2 fluorescence is plotted versus side scatter (SSC). Dead cells and debris are excluded from the analysis.
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91
Fig. 3. The principle of multiparameter high gradient magnetic cell sorting (MACS-MultiSort). After a first separation (as illustrated in Fig. 1), magnetic MicroBeads are released from the cell surface by enzymatic treatment (A). Cells are magnetically labeled for a second parameter and applied on a new separation column (B). Only cells expressing the second marker are retained in the magnetic field; non-labeled cells are isolated as negative (depleted) fraction (C). After removal of the column from the magnetic field labeled cells are eluted (D).
isolation of rare cells and the processing of large cell numbers.
3. Method The magnetic label is provided by superparamagnetic beads conjugated to specific ligands or antibodies. Cells are separated on ferromagnetic matrices in a high-gradient magnetic field. Nonmagnetic cells pass this matrix, whereas magnetically tagged cells are retained and subsequently eluted after removal of the column from the magnetic field (Fig. 1). This technology allows the processing of cell numbers above 1010 in a short time.
4. Purity, viability and functional potential of isolated cells Not only can high purities be achieved, cells are also separated in a gentle way. No strong mechanical forces influence cell integrity or viability. Because all cells are sorted simultaneously, up to 1011
cells can be processed in about 30 min, giving this method a leading edge in the sorting of rare cells. The little physical stress during MACS sorting favors the recovery of viable cells, unlike FACSsorting, where the cells are submitted to considerable stress by acceleration in the nozzle. Magnetically sorted cells have been used to generate dendritic cells from CD14 + monocytes [9], to analyse cytokine secretion of T-cell subsets and the cytokine secretion patterns of CD61 expressing megakaryocyte progenitors [10], to investigate Tcell–B-cell interactions in complex in vitro culture systems [11,12], to expand human haematopoietic progenitor cells ex vivo [13] and even for cell transfer experiments [14]. A recent development is a clinical grade separation system which performs the separation of CD34 expressing human haematopoietic progenitor cells from various sources and will be used in cellular therapy of human malignant or inherited diseases [15].
5. Isolation of rare cells The potential of high gradient magnetic cell
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Fig. 4. Isolation of CD4 + CD45RO − CD45RA + T-cells from human PBMC using multiparameter magnetic cell sorting. Cells are stained with CD4 PE (A, B) or CD45RO PE and CD45RA FITC (C, D). A: PBMC before CD4 separation. B: positive fraction after MACS enrichment of CD4 + Th cells. C: after removal of CD4 specific MicroBeads cells are stained with CD45RO specific MicroBeads, PE conjugated CD45RO and FITC conjugated CD45RA FITC. D: negative fraction after MACS depletion of CD45RO + cells.
sorting is best recognised when sorting of rare cells is desired. Here, if the frequency of positive cells becomes very low ( B 0.1 – 1%), highly efficient labeling of the desired cell population paired with a short processing time is a prereq-
uisite of successful isolation. CD34 expressing haematopoietic progenitor cells in normal human peripheral blood represent only 0.1% of peripheral mononuclear cells (PBMC). However, the MACS-technology allows an efficient isola-
A. Thiel et al. / Immunotechnology 4 (1998) 89–96
93
Fig. 5. Isolation of CD34 + HLA–DR − cells from human PBMC using multiparameter magnetic cell sorting. Cells are stained with CD34 PE and HLA – DR FITC. A: MACS isolated CD34 + cells before MACS depletion of HLA – DR − CD34 + cells. B: negative fraction after depletion of HLA–DR − CD34 + cells. (The frequency of CD34 + cells among PBMC is about 0.1%; thus HLA – DR − CD34 + cells represent about 0.001% of total PBMC).
tion of this cell population (Fig. 2). Functional tests reveal excellent viability of the isolated cells [13,16]. For the isolation of CD34 + cells from various tissues and from peripheral blood of normal donors no other method is available that achieves comparable enrichment rates (up to 20000-fold) and recoveries [17 – 19]. Allergen-specific B-cells from the peripheral blood of atopic donors (1 in 10 − 4) [20] and fetal cells from the peripheral blood of pregnant woman (1 in 10 − 5) [21,22] have been isolated successfully. Recently a method has been developed for the enrichment of tumor cells in peripheral blood, decreasing the detection limit for residual cancer cells in tumor patients [30].
A new challenge for cell separation is the isolation of transfected cells according to surface marker expression. Conventional selection of transfected cells by cotransfection with drug resistance genes (Gregor Siebenkotten, personal communication) is a very time consuming procedure. Recent studies suggest that magnetic cell sorting is in fact a powerful strategy for the isolation of transfected cells. Cells are cotransfected with the vector of interest and an expression vector for surface molecules, which can be specifically labeled with magnetic microparticles. At defined timepoints after transfection, cells expressing the marker molecule can be isolated, even if the rate of transfection is low. Separated cells can directly be analysed functionally, thereby eliminating the
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influence of growth advantages of cells that are not transfected (Gregor Siebenkotten, personal communication).
Fig. 6. The principle of the ‘affinity-matrix-technology’. Secreted products are ‘caught’ on the cell surface by the affinity matrix created by ‘catch’ antibodies and subsequently detected by labeling with a second ‘detection antibody’ conjugated to MicroBeads or fluorochromes. Fig. 8. Principle of signal enhancement by magnetofluorescent liposomes. A: conventional reagents bind only 1 – 10 fluorochromes per antibody. B: liposomes conjugated to specific antibodies are filled with several thousand fluorescein molecules which drastically increases the fluorescence signal intensity of labeled cells compared to conventional antibody conjugates.
6. Multiparameter high gradient magnetic cell sorting
Fig. 7. Magnetic cell separation of murine Th splenocytes according to secreted IL-2. Cells are stained for secreted IL-2 caught by the affinity matrix. A: polyclonally activated murine spleen cells before IL-2 separation. B: positive fraction of MACS separated IL-2 producing murine splenocytes.
The recent development of multiparameter magnetic cell sorting (MACS-MultiSort) eliminated the last drawback of magnetic cell sorting, namely the restriction to a single parameter [8]. After the first step of isolation, the primary magnetic labeling is removed enzymatically. A secondary magnetic labeling can now be performed as illustrated in Fig. 3. Fig. 4 shows an example of multiparameter high gradient magnetic cell sorting. The CD45RO − CD45RA + subset of CD4 + Th cells is efficiently separated from CD45RO + CD45RA − CD4 Th cells. Multiparameter sorting allows also the isolation of extremely rare subsets of cells. For example, uncommitted subsets of human haematopoietic progenitor cells represent
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95
Fig. 9. Staining of IL-6 receptor: conventional staining versus magnetofluorescent liposomes. Purified CD34 + cells are stained with CD34 Cy-Chrome and A: anti IL-6R PE or B: anti IL-6R DIG + anti DIG liposomes.
extremely rare cell populations. Only three cells in 105 PBMC are characterised by CD34 expression and lack of HLA–DR expression. The enrichment of such cells from an initial frequency of 0.003 – 96% (Fig. 5) stands for an 800000-fold enrichment rate.
products for a short time in vitro, and the secreted product is caught on the surface by the affinity matrix. It can then be labeled and analysed with fluorochrome or magnetically tagged antibodies. Using this technology, antibody secreting cells or activated, IL-2 (Fig. 7) or IFN-g producing T-cells have been isolated and characterised functionally [26].
7. New parameters
7.2. Magnetofluorescent liposomes Immunofluorescent analysis and sorting of live cells can be limited: either suitable surface markers are expressed only in low density or the antigen of interest is not expressed on the cell surface, e.g. secreted molecules like cytokines, only allowing analysis in fixed cells after permeabilization [23]. Recently, new technologies have been developed to overcome these limitations for analytical and preparative cytometry.
7.1. Sorting of li6e cells according to secreted products The first method for the analysis and isolation of live cells according to their secreted products was published by our group [24]. We have recently shown that this method can easily be adapted to the analysis and separation of cells secreting antibodies, cytokines or growth factors [25]. The labeling of cells according to the products they secrete can be performed by using the Biotin – Avidin-system to attach an affinity matrix onto the cell surface (Fig. 6). The cells are allowed to secrete
The detection limit of conventional immunofluorescence is in the range of several thousand molecules per cell depending on cellular autofluorescence [27]. Therefore many functionally important molecules, e.g. receptors for cytokines, hormones or growth factors were difficult to analyse, because most of them are expressed in low copy numbers. We have developed a sensitive reagent, magnetofluorescent liposomes, conjugated to specific antibodies which increase fluorescence signal intensity up to 1000-fold (Figs. 8 and 9) [28]. Thus magnetofluorescent liposomes allow clear detection of cell subsets expressing 300–400 antigens or less. In addition, they offer the possibility of sorting out labeled cells magnetically. Liposomes have been used to demonstrate for the first time the specific expression of IFN-g on the surface of IFN-g producing Th1 cells [29]. It was also possible to identify and separate functional distinct subpopulations of CD34 + hematopoietic stem cells according to the expression of receptors for IL-6 (A. Thiel, manuscript in preparation).
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8. Summary Separation of specific cell populations is a prerequisite for many analytical and functional studies in basic research as well as for diagnostic and therapeutic applications. The development of high gradient magnetic cell sorting during the last years offers a highly specific, gentle and fast way of purification of a large panel of different cell types from complex cell mixtures. Standard separations can be done within minutes using either positive or negative enrichment strategies. The magnetic particles influence neither viability or function of labeled cells nor analytical parameters of flow-cytometry. The high specificity of magnetic labeling and the short separation time result in fast processing of high cell numbers therefore allowing the purification of even rare cell populations within 1 – 2 h. In addition, the new technologies described here, e.g. magnetofluorescent liposomes for the detection of rare antigens, the ‘affinity matrix technology’ to detect secreted molecules and multiparameter magnetic cell sorting raise the number of parameters which can be used for identification and separation of specific cell populations.
Acknowledgements Many thanks to Florian Kern and Christine Raulfs for critically reading the manuscript. This work was supported by a grant from the ‘Deutsche Krebshilfe e.V.’ (W8/94/RA1).
References [1] Radbruch A, editor. Flow cytometry and cell sorting, Heidelberg: Springer, 1992. [2] Silvestri FF, Banavali SD, Hulette BC, Civin CI, Preisler HD. Int J Cell Cloning 1991;9:474–90. [3] Smeland EB, Funderud S, Kvalheim G, Gaudernack G, Rasmussen AM, Rusten L, Wang MY, Tindle RW, Blomhoff HK, Egeland T. Leukemia 1992;6:845–52. [4] Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D, Knitter G, Buckner CD, Bernstein ID. J Clin Invest 1988;81:951 – 5. [5] Molday RS, Molday LL. FEBS Lett 1984;170:232–8. [6] Miltenyi S, Mu¨ller W, Weichel W, Radbruch A. Cytometry 1990;11:231 – 8. .
[7] Radbruch A, Mechtold B, Thiel A, Miltenyi S, Pfluger E. In: Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Methods Cell Biology. San Diego: Academic Press, 1997:387 – 402. [8] Thiel A, Schmitz J, Miltenyi S, Radbruch A. Immunol Lett 1997;57:189 – 92. [9] Pickl WF, Majdic O, Kohl P, Stockl J, Riedl E, Scheinecker C, Bello-Fernandez C, Knapp W. J Immunol 1996;157:3850– 9. [10] Schmitz B, Radbruch A, Ku¨mmel T, Wickenhauser C, Korb H, Hansmann ML, Thiele J, Fischer R. Eur J Haematol 1994;52:267 – 75. [11] Blanchard D, Gaillard C, Hermann P, Banchereau J. Eur J Immunol 1994;24:330 – 5. [12] Defrance T, Vanbervliet B, Briere F, Durand I, Rousset F, Banchereau J. J Exp Med 1992;175:671 – 82. [13] Servida F, Soligo D, Caneva L, Bertolini F, de Harven E, Campiglio S, Corsini C, Deliliers GL. Stem Cells 1996;14:430 – 8. [14] Schmitz J, Thiel A, Kuhn R, Rajewsky K, Muller W, Assenmacher M, Radbruch A. J Exp Med 1994;179:1349– 53. [15] McNiece I, Briddell R, Stoney G, Kern B, Zilm K, Recktenwald D, Miltenyi S. J Hematother 1997;6:5 – 11. [16] de Wynter EA, Coutinho LH, Pei X, Marsh JC, Hows J, Luft T, Testa NG. Stem Cells (Dayt) 1995;13:524 – 32. [17] Kato K, Radbruch A. Cytometry 1993;14:384 – 92. [18] Radbruch A, Mechtold B, Thiel A, Miltenyi S, Pfluger E. In: Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Methods Cell Biology. San Diego: Academic Press, 1997:387 – 402. [19] Thiele J, Wickenhauser C, Baldus SE, Kuemmel T, Zierbes T, Drebber U, Wirtz R, Thiel A, Hansmann ML, Fischer R. Leuk Lymphoma 1995;16:483 – 91. [20] Irsch J, Hunzelmann N, Tesch H, Merk H, Maggi E, Ruffilli A, Radbruch A. Immunotechnology 1996;1:115 – 25. [21] Busch J, Huber P, Pfluger E, Miltenyi S, Holtz J, Radbruch A. Prenatal Diagn 1994;14:1129 – 40. [22] Ganshirt-Ahlert D, Borjesson-Stoll R, Burschyk M, Dohr A, Garritsen HS, Helmer E, Miny P, Velasco M, Walde C, Patterson D, et al. Am J Reprod Immunol 1993;30:194 – 201. [23] Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. J Immunol Methods 1993;166:201 – 14. [24] Manz R, Assenmacher M, Pflu¨ger E, Miltenyi S, Radbruch A. Proc Natl Acad Sci 1995;92:1915 – 21. [25] Manz RA, Thiel A, Radbruch A. Nature 1997;388:133 – 4. [26] Assemacher M, Lo¨hning M, Scheffold A, Manz A, Schmitz J, Radbruch A. Eur J Immunol 1998;28:1534 – 43. [27] Scheffold A, Miltenyi S, Radbruch A. Immunotechnology 1996;1:127 – 37. [28] Assenmacher M, Scheffold A, Schmitz J, Segura Checa J A, Miltenyi S, Radbruch A. Eur J. Immunol 1996;26:263 – 7. [29] Assenmacher M, Scheffold A, Schmitz J, Checa JS, Miltenyi S, Radruch A. Eur J Immunol 1996;26:263 – 7. [30] Martin V, Siewert C, Scharl A, Harms T, Heinze R, O8 hl S, Radruch A, Miltenyi A, Schmitz J. J Exp Hematol1998;26:252– 64.