WO2010129441A2

WO2010129441A2 - Method for separating stem cells from their more differentiated progeny using microfluidic devices

Info

Publication number: WO2010129441A2
Application number: PCT/US2010/033298
Authority: WO
Inventors: Michael Grisham
Original assignee: Gpb Scientific, Llc
Priority date: 2009-05-04
Filing date: 2010-04-30
Publication date: 2010-11-11
Also published as: WO2010129441A3

Abstract

The technology described herein relates to methods and devices for separating cells. The methods and apparatus permit the removal of differentiated, or more committed, cells from stem cell cultures.

Description

Method for Separating Stem Cells from their More Differentiated Progeny Using Microfluidic Devices

Cross Reference to Related Applications

The present application claims the benefit of United States provisional application US 61/175,418, filed on May 4, 2009, the contents of which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to methods and devices for preparing and maintaining populations of stem cells.

Background of the Invention

The identification of stem cells, and particularly totipotent, pluripotent, and omnipotent cells, represents a major advance in biology with extraordinary potential in areas of tissue engineering and cell based therapies. One challenge facing stem cell research is that of maintaining populations of cells in a state of differentiation that allows the cells to be guided to differentiate along controlled lineages at the desired time. As stem cells grow in the laboratory, they tend to differentiate in an uncontrolled manner, and the resulting mixture of cells must be purified if the parent culture is to be effectively maintained.

The separation of materials by size or mass is a fundamental analytical and preparative technique in biology, medicine, chemistry, and industry. Separation of particles, and particularly cells, by size has been accomplished by a variety of means including cell sieves having fixed pore sizes and through the use of fixed arrays of obstacles (micro/nano- fabricated structures) in a flow path of a microfluidic device. Among the microfluidic devices described in the art as useful for cell separation are those found in U.S. Patent No. 7,150,812 (see also US 5,715,946; US 2007/0187250; US 2008/0023399; US 6,632,652;

US 5,837,115; US 5,427,663; US 7,318,902; US 7,276,170; US 7,472,794; US

2009/0188795; US 6,685,842; US 6,913,697). These devices provide a rapid means for segregating a large population of cells based upon size. Summary of the Invention

Described herein are methods and devices for the separation of stem cells from their differentiated progeny. Such methods and devices permit the maintenance of stem cells in a desired state of differentiation and in a state where they are free, or substantially free, of differentiated cells. Cultures that are "substantially free" of differentiated cells have less than 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1%, 0.5%, 0.2% or 0.1 % of the total cells in different states of differentiation, with lower percentages being preferred.

The methods and apparatus described herein can be used to separate a relatively large number of cells on a basis that is discrete (single pass separation of a population of cells) or continuous (separation of cells, especially rare cells, from a population of cells and return of some portion of the cells back to the initial population). In addition, the apparatus and methods provide a separation procedure that is rapid, capable of segregating rare cells from a large population of cells, and gentle enough to avoid physically injuring cells or inducing stem cell differentiation. Size separations may be combined with separation methods based upon additional characteristics, including the presence of cell surface antigens.

In a first aspect, the invention is directed to a method of treating a population of stem cells to enrich it in cells at a specific state of differentiation. This is accomplished by introducing a first population of stem cells into one or more microfluidic channels of a microfluidic device wherein the cells are in the form of a suspension of cultured cells. This population includes stem cells in a first differentiation state (A), having an average diameter x, and stem cells of the same lineage that are in a second differentiation state (B), having an average diameter y that is different from x. For example, the stem cells in differentiation state A may be totipotent (i.e., in the least differentiated state and capable of forming embryonic and extraembryonic cell types) and these cells may differentiate into B cells that are unipotent (capable of developing into only a single cell type) or they may fully differentiate (e.g., into a myocyte). For the purposes herein, cell type A and cell type B are of the same lineage if they are developmentally related such that, either directly or through one or more intermediate cell types, A can give rise to B or vice versa. The microfluidic channels of devices have gaps, pores or spaces between obstacles that have a diameter effective for separating cells of diameter x from cells of diameter y. In general, this means that the first population of cells will be enriched in cells at a specific differentiation stage by at least 10%, with higher percentages, e.g., at least 20, 40 or 60%, being preferred. Although not a requirement for separation, the gaps, pores or spaces present in microchannels will usually have an average diameter between x and y.

Once the stem cells have been introduced into a device, they are propelled thorough the microfluidic channels to one or more outlets, resulting in the separation of A type cells from B type cells. The cells are then collected in separate aliquots based upon size. For example, one outlet may be positioned so as to receive a stream of cells that has been diverted to that position based upon having a size that penetrates gaps or pores in a microfluidic channel whereas another outlet may be positioned to receive cells that have been diverted to that location based upon their having a diameter that is excluded by gaps or pores in the microfluidic channel. Relative to the first population of stem cells, this "second population" of stem cells has been enriched in cells in either the first (A) or said second (B) differentiation state.

Types of stem cells that can be enriched by this process include totipotent, pluripotent, multipotent, oligopotent or unipotent stem cells. In one embodiment, the microfluidic device may include a first array comprising a network of gaps within the microfluidic channels and the stem cells may be propelled through these channels by a field.

This field may be the result of fluid flow, centrifugal, gravitational, hydrodynamic, pressure gradient, or capillary action. A flux of the field from the gaps is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field. Cells having a size less than a predetermined first critical size, generally between x and y, are transported in the average direction of the field, and cells having a size at least that of the first critical size are transported generally in the average direction of the major flux component. The term "generally," in this context refers to more than 50% of the cells. Thus, cells are separated according to size. In another embodiment, the microfluidic device comprises an ordered first array of obstacles within the microfluidic channels. These obstacles are asymmetric with respect to the average direction of the field such that, when cells are introduced into the first array, those having a size less than a predetermined first critical size between x and y are transported in a first direction, and cells having a size at least that of the first critical size are transported in a second direction. Since the first and second directions are different, this leads to a separation of cells according to size.

The devices described above may include a second array of obstacles within the microfluidic channels, this second array being fluidically coupled to, and downstream from, the first array. The second array may comprise a network of gaps or pores between obstacles that are of a different diameter than the gaps or pores in the first array. When stem cells are propelled through the first and second arrays by a field, a flux of the field from the gaps of the second array is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field. Cells introduced into the second array that have a size less than a predetermined critical size of the second array are transported generally in the direction of the field, and cells having a size at least that of the critical size of the second array are transported generally in the direction of the major flux component.

The microfluidic devices may also include one or more regions or obstacles that have a substance that preferentially binds to one type of cell, a magnetic agent bound to the surface of one type of cell, or a non-magnetic agent bound to the surface of one type of cell. Preferential binding, in this context, means binding one type of cell in a population of stem cells to a greater extent than to other types of cells in the population. For example, the substance may selectively bind to a protein or antigen found on the surface of an embryonic stem cell or adult stem cell as compared to differentiated cells arising from the embryonic or adult stem cells. Preferably, the substance is a monospecific polyclonal antibody, monoclonal antibody, scFv, Fab fragment, a Fab' fragment, a F(ab')₂, Fv, or a disulfide linked Fv directed against antigen. Preferred antigens are CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD105 CD349, CD140b, CD324; CD44, CD29, HLA-A,B,C, CD13, CD166, CD49e, CD2711ow, CDlO, CD14, CDl 17, CD133, HLA-DR and SSEA-4. In any of the methods described above, the second population of stem cells collected after size separation may be tested for one or more cell surface stem cell markers. The preferred markers are CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD 105 CD349,

CD140b, CD324; CD44, CD29, HLA-A,B,C, CD13, CD166, CD49e, CD2711ow, CDlO, CD14, CDl 17, CD133, HLA-DR and SSEA-4.

In another aspect, the invention is directed to an apparatus for the culture or propagation of cells having a chamber for maintaining cells in suspension and a microfluidic device having an inlet for receiving cells from the chamber. Thus, the inlet of the microfluidic device is in fluid connection with chamber. The phrase "cells in suspension" refers to cells in a state where they do not adhere tightly to the walls of the chamber and can be introduced into the microfluidic device substantially as a population of single cells (i.e., at least 50, 70 or 90% of the cells should be single cells). The device has at least one microfluidic channel with a first array comprising a network of gaps or pores and a first outlet for receiving cells having a size less than a first critical size. The device also has a second outlet for receiving cells from the array wherein the cells have a size at least that of the first critical size. Either the first outlet or the second outlet is in fluid connection with the chamber, thereby permitting cells to be returned to the chamber. Thus, in some embodiments, cells having a size less than the first critical size may be returned to the chamber and, in other embodiments, cells having a size at least that of the first critical size may be returned to the chamber.

A field is used to propel the cells through the microfluidic channel and the flux of the field from the gaps is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field. When cells are introduced into the first array, those having a size less than a first critical size are transported generally in the average direction of the field, and cells having a size at least that of the first critical size are transported generally in the average direction of the major flux component, thereby separating the cells according to size.

In some embodiments, the apparatus for the culture or propagation of cells has a first array and a second array. The second array comprises a network of gaps within the microfluidic channel and, in the presence of a field that propels the cells through the microfluidic channel, the second array receives a flow of cells from the first array. The flux of the field from the gaps of the second array is divided unequally into a second major flux component and a second minor flux component such that the average direction of the second major flux component is not parallel to the average direction of the field of the second array. When introduced into the second array, cells having a size less than a second critical size are transported generally in the average direction of the field, and cells having a size at least that of the second critical size are transported generally in the average direction of the second major flux component, thereby separating the cells received from said first array according to size.

In another aspect, devices may be used to separate other cells of a common lineage based upon size differences. For example, a preparation enriched in mitotic cells may be made using essentially the same size-based separation techniques described above. In this case, a suspension of cultured cells, comprising a fraction of cells undergoing mitosis, is introduced into one or more microfluidic channels of a microfluidic device. These channels have gaps, pores or obstacle spaces with a diameter effective for separating mitotic cells from non-mitotic cells on the basis of size. The population of cells is propelled through the microfluidic channels to one or more outlets where a second population of cells, now enriched in cells undergoing mitosis, is collected.

In one embodiment, the microfluidic device comprises a first array comprising a network of gaps within the microfluidic channels. Cells are propelled through the microfluidic channels by a field and a flux of the field from the gaps in the first array is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field. When cells are introduced into the first array, those having a size less than a predetermined first critical size between the diameter of mitotic and nonmitotic cells are transported generally in the average direction of the field, and cells having a size at least that of the first critical size are transported generally in the average direction of the major flux component, thereby separating the cells according to size. The method may also use a device having an ordered first array of obstacles that is asymmetric with respect to the average direction of the field, such that, when cells are introduced into the first array, cells having a size less than a predetermined first critical size are transported in a first direction, and cells having a size at least that of the first critical size are transported in a second direction, wherein said first and second directions are different. This results in the separation of cells according to size.

In preferred embodiments, the population of cells that comprise a fraction of cells undergoing mitosis is a clonal culture comprising a single cell type or a synchronized cell culture. Synchronization may be achieved by treating the population of cells with an inhibitor of mitosis such as colchicine, vinblastin or nocodazole.

Brief Description of the Drawings

FIG. 1 : Panel A shows a schematic diagram of an array device. The device consists of an obstacle array asymmetric about the field direction. Panel B shows a schematic diagram of a perspective view of a preferred array, comprising a base substrate with a main surface, on which an obstacle course is made to form a network of gaps, wherein cascades of unequal bifurcations of field flux occur. The obstacle course may be sealed to a cap layer to form an enclosed network of gaps.

FIG. 2 shows a schematic diagram of an obstacle array. The obstacle array is symmetric about the dotted lines (principle axis), but asymmetric with respect to the field direction.

FIG. 3 shows a schematic diagram of a network of gaps defined by an array of obstacles. Each streamline represents an equal amount of field flux. The field flux from one gap is divided into two subsequent gaps, wherein the amounts of flux going into the two gaps are unequal. In this case, two streamlines goes to the left (major flux component) and one streamline goes to the right (minor flux component) at each gap. The array direction is indicated by the gray arrow. FIG. 4 shows a schematic diagram of particles separated in an array device according to an embodiment of the invention. The small particles move along field and large ones towards array direction.

FIG. 5 shows a schematic diagram of an obstacle array, showing characteristic array dimensions, λ denotes the period of a row of obstacles, d the gap spacing between obstacles, and αλ the lateral shift of every row.

FIG. 6 shows a schematic diagram of a particular array, showing that field lines go around obstacles. The dotted line at the center marks the division of field lines going to different sides of the obstacle.

FIG. 7 shows a schematic diagram of a particular array illustrating the displacement of large particles by obstacles.

FIG. 8 shows a plot of particle migration direction versus size. There exists a critical particle size Ro where a sharp transition of migration direction occurs.

FIG. 9 shows a schematic diagram of an apparatus for the culture of cells, such as stem cells, having two arrays of different critical sizes in series. Particles at output (outlet) 1 are smaller than Rl, the critical size of the first array in the series, those at output (outlet) 2 between size Rl and R2, and the ones at output 3 are larger than R2, the critical size of the second array. The apparatus is shown having a chamber for maintaining cells in fluid connection with the inlet of the microfluidic array, and the output of cells having a size between Rl (first critical size) and R2 (second critical size) from outlet 2 being returned to the chamber via the Return Path, which is shown by the series of dashed arrows. The small dash circles show some exemplary positions where one or more regions or obstacles comprising a substance that preferentially binds to one type of cell can be placed.

Alternatively, a substance that preferentially binds to one type of cell can be applied over portions or all of the array so that size and affinity separation can be accomplished simultaneously. Additional inlets into the array and chamber for fluid media are not shown. FIG. 10 shows a schematic diagram of multiple arrays in series, wherein each array has a different critical size. The dotted horizontal line represents the division between subsequent arrays and each subsequent array in the series has an increasing critical size. A continuous distribution of molecular sizes can thus be fractionated and analyzed with one device.

FIG. 11 shows a schematic top view of an embodiment of the invention, using a square array of circular obstacles to form the network of gaps, tilted at a small angle θ with respect to the field. The square array is spanned by primitive vectors x and y, which are perpendicular to each other. Bifurcation of field flux occurs because of the asymmetry of the obstacle array with respect to the field.

FIG. 12 shows a schematic diagram of a device used to concentrate sample. The field direction can be defined by the sidewalls of the array. The shaded area shows the trajectories of particles larger than the critical size, injected from the top. Particles move against the sidewall and are concentrated.

FIG. 13 shows a schematic diagram of a device having an ordered array of obstacles and employing a non-uniform field, wherein the direction of the field changes across the array.

FIG. 14 shows a schematic diagram of a device having an ordered array of obstacles in a curved microfluidic channel. The curved channel may result in a field that is nonuniform.

Definitions

The term "channel" as used herein refers to a structure in which fluid may flow. A channel may be a capillary, a conduit, a strip of hydrophilic pattern on an otherwise hydrophobic surface wherein aqueous fluids are confined, etc.

The term "microfluidic" as used herein, refers to a system or device having one or more fluidic channels, conduits or chambers that are generally fabricated at the millimeter to nanometer scale, e.g., typically having at least one cross-sectional dimension in the range of from about 10 nm to about 1 mm. As used herein, "cells in suspension" means cells in a state where they do not adhere tightly to the walls of the vessel (e.g., chamber) in which they are being grown or maintained. In cases where a cell type is grown as an adherent cell it can be treated to provide cells in suspension by the use of various agents such as trypsin. When in suspension, the majority of cells present can be introduced into a microfluidic device for separation by size, affinity etc. In some embodiments, the majority, or greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of the cells in suspension are present as single cells, with higher percentages being preferred.

As used herein "stem cell" or "stem cells" are cells that have the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues. The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. Stem cells can be totipotent, pluripotent, multipotent, oligopotent or unipotent.

Totipotent (omnipotent) cells can differentiate into embryonic and extraembryonic cell types. Such cells can develop into a complete, viable, organism. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, e.g., cells derived from any of the three germ layers. Multipotent stem cells are cells that can differentiate into a number of cells, but only those of a closely related family of cells. Oligopotent stem cells can differentiate into only a few cell types, such as lymphoid or myeloid cells. Unipotent cells are stem cells that can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells {e.g., muscle stem cells).

As used herein "a population of cells enriched in specific stem cells" means a population of stem cells that are enriched in stem cells a specific stage of differentiation. Thus, a population of stem cells that is subject to the methods described herein to remove cells that are precursors of the stem cells (e.g., removal of totipotent cells from a culture of pluripotent cells) or to remove cells that have differentiated from the population of stem cells in the desired stage of differentiation (e.g., removal of terminally differentiated cells or unipotent cells from multipotent cells) results in a population of cells enriched in specific stem cells. As used herein the term "field" refers to any force or vector entity that can be used to propel cells through an array or arrays. The field that drives the particles being separated may be a force field, such as an electric field, a centrifugal field, or a gravitational field. The field may also be a fluid flow, such as a pressure-driven fluid flow, an electro-osmotic flow, capillary action, etc., wherein the vector entity is the fluid flux density. Further, the field may be a combination of a force field and a fluid flow, such as an electrokinetic flow, which is a combination of the electric field and the electro-osmotic flow. In some embodiments, the average direction of the field will be parallel to the walls of the channel that contains the array.

"Array" as used herein is a series of obstacles in a channel that cause the separation of cells based one or more properties {e.g., size). In some embodiments, particularly those where cells are separated based upon size using a microfluidic device such as described in U.S. 7,150,812, an array comprises a network of gaps that creates a field pattern such that the field flux from a gap within the network is divided into unequal amounts (a major flux component and a minor flux component) into the subsequent gaps (for example, see FIG. 3), even though the gaps may be identical in dimensions. Looking at such an array as a whole, on average, the unequal divisions of the field flux are weighted in one direction. Thus, the major flux components are diverted, on average, in the same direction (i.e., diverted to the same side of the obstacle), such that the average direction of the major flux components is not parallel to the average direction of the minor flux components, or to the average direction of the field. It is preferred that a majority of the major flux components be diverted in the same direction. In a particularly preferred embodiment, the array is an ordered array, wherein the major flux component from each bifurcation event is diverted in the same direction. Generally, the minor flux component exiting one gap feeds into the major flux component exiting a subsequent gap (FIG. 3). The terms "gap," "obstacle," "asymmetric," "ordered," "total field flux" and the "offset angle " are as defined in U.S. Patent 7,150,812.

The term "critical size" as used herein refers to a diameter of gaps, pores or spaces present in a microfluidic device that is effective in the separation of different cell types. For example, if totipotent stem cells are being separated from terminally differentiated cells, then a critical size would be one that accomplished this separation for a particular device. A critical size will often, but not necessarily, be intermediate between the diameter of the cell types being separated. It will be recognized that there will be more than a single critical size for a separation and that what constitutes a critical size will be determined, in part, by the types of cells being separated and the type of device being used. It will also be recognized that there will be some variation among the gaps, pores and spaces present in a device. Critical sizes can be empirically determined by using a series of devices with different average gap, pore or space diameters and examining the samples collected from one or more outlets on the devices to determine whether a separation of cell types has been achieved.

Detailed Description of the Invention

The technology described herein provides for the preparation and maintenance of stem cell cultures, isolation of cells at various stages of the cell cycle, and apparatus for use in those methods. The apparatus employ a microfluidic device for separating cells according to size using an array or arrays of obstacles. In some embodiments apparatus may employ a microfluidic device comprising one or more arrays of obstacles as described by Huang et al. in U.S. 7,150,812, which is incorporated herein by reference in its entirety. Such arrays comprise a network of gaps, and a field is applied to the arrays to propel the particles being separated through the array.

In one embodiment, the array is an ordered group of obstacles in a channel, which is asymmetric with respect to the direction of the applied field. In other embodiments, the methods and apparatus described herein can employ microfluidic devices or arrays, as described in U.S. Pat. Nos. 6,685,841 or 6,913,697, or as described by Toner and his colleagues in U.S. Pub. Nos.: 2007/0259424; 20070160503; 2005/0266433; 2007/0099207; 2007/0059774; and 2008/0113358 for the selection of cell populations. Other published applications that are of interest include: US 2006/0134599; US 2005/0282293; US 2006/0121624; US 2007/0196820; US 2007/0059716; US 2007/0059680; US US 2007/0059718; US 2007/0059719; US 2007/0059781; US 2007/0026413; US 2007/0026414; US 2007/0026415; US 2007/0026417; US 2006/223178; US 2007/0026418; US 2007/0026381; US 2008/0090239; US 2008/0124721; US 2008/0138809; US 2008/0220422; US 2008/0113358; US 2009/0181421; US 2007/0172903; US 2007/0231851; US 2007/0264675; US 2010/0055758. Each of these patents and publications are herein incorporated by reference in their entirety. In cases where more than one array or region is employed in a microfluidic device to separate and/or purify stem cells the microfluidic devices may employ a combination of the microfluidic devices or arrays set forth in the above publications and in U.S. 7,150,812. Such combinations include simultaneous size and affinity (e.g., immunoaffϊnity) selection and purification of cells (e.g., maintenance of pluripotent or multipotent stems cell cultures by removal of differentiated cells).

Due to the efficiency of microfluidic devices such as those described above, and the ability to gently separate large numbers of cells having different sizes, it is possible to separate cells without damage or trauma. The ability to separate cells based on size also permits separation of stem cells from cells that have differentiated from those stem cells provided they have a different size. The incorporation of regions or obstacles comprising a substance that preferentially binds to one type of cell, permits further selection or purification of stem cell cultures when size alone in insufficient. This may be accomplished by utilizing a substance that preferentially binds to an antigen that appears on the surface of the differentiated cells or on the surface of the stem cells.

In addition to purifying a population of stem cells by removing differentiated cells, the differentiated cells may also be isolated or collected using such methods. In this way, using a cultured cell type that is less differentiated (e.g., pluripotent or totipotent), it is possible to isolate a population of cells comprising more highly differentiated cells (e.g., multipotent or unipotent) or fully differentiated cells or a combination thereof.

The methods and apparatus of the present invention offer a variety of advantages, such as the ability to separate stem cells from differentiated cells while having a limited effect on the differentiation of stem cells. In addition, the apparatus may serve as a high throughput sample preparation tool for maintaining stem cell cultures free of contaminating differentiated cells.

A. Methods

The methods and apparatus described herein may be used in the field of cell biology and related fields by permitting the separation and purification of cells, particularly stem cells or cells undergoing division. In one embodiment, the microfluidic device comprises a microfluidic channel and an array comprising a network of gaps within the microfluidic channel. A field is used to propel the cells being separated through the microfluidic channel and, during this process, the individual field flux exiting a gap is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field.

In another embodiment, the methods and apparatus provided for herein employ a microfluidic device for separating cells according to size comprising a microfluidic channel, and an ordered array of obstacles within the microfluidic channel. The ordered array of obstacles is asymmetric with respect to the average direction of the applied field.

Another embodiment of the methods and apparatus described herein provides for the use of a microfluidic device for separating cells according to size comprising a microfluidic channel, and multiple arrays in series within the microfluidic channel, wherein each array has a different critical size. Each of the arrays comprises a network of gaps wherein a flux of the field from the gaps is divided unequally into a major flux component and a minor flux component into subsequent gaps in the network. The average direction of the major flux components in each array is not parallel to the average direction of the field.

B. Stem Cell Separation

Stem cells may be grown and induced to differentiate into different cell types using methods known in the art, including those described in Placzek et ah, J R. Soc. Interface 6:209-232 (2009), Parolini et al, Stem Cells 26:300-311 (2008) and references cited therein. However, cultures of stem cells, regardless of whether the cultures are totipotent, pluripotent, multipotent, oligopotent, or unipotent, have a tendency to produce differentiated progeny. In order to maintain and expand cultures of stem cells, it is necessary to separate cells at a desired stage of differentiation from cells at other stages.

In one procedure, a first population of stem cells is enriched in cells at a desired stage of differentiation by introducing the cells into a microfluidic device for separating cells according to size. The microfluidic device has at least one microfluidic channel with a first array of gaps, A field is used to propel the cells being separated through the microfluidic channel and a flux of the field from the gaps is divided unequally into a major flux component and a minor flux component that continues on to subsequent gaps. The average direction of the major flux component is not parallel to the average direction of the field and when cells are introduced into the array, cells having a size less than a first critical size are transported generally in the average direction of the field and cells having a size at least that of the first critical size are transported generally in the average direction of the major flux component, thereby achieving a separation. Finally, stem cells of a desired size are collected from an outlet on the device.

In a second enrichment procedure, a first population of stem cells is enriched in cells at a desired stage of differentiation using a device with a microfluidic channel having an ordered first array of obstacles which is asymmetric with respect to the average direction of the field used to propel cells. When cells are introduced into the array, those having a size less than a first critical size are transported in a first direction, and cells having a size at least that of the first critical size are transported in a second, different direction. Again, stem cells of a desired size are collected from an outlet on the device.

In one embodiment of the foregoing enrichment procedures, a stem cell population of a desired size that has been collected from an outlet on a microfluidic device is reintroduced into the first population of stem cells, i.e. the enriched stem cells are recycled back to the population being applied to the device. This method may be carried out substantially continuously so that cells of an unwanted differentiation state are removed from the first population of stem cells. The method may also be used to remove differentiated cells from the first population of stem cells by affinity (discussed further below) or by a combination of affinity and size.

By "substantially continuously," it is meant that the method is carried out over sufficient time to reduce the number differentiated cells in the first population by at least 30%, 40% 50%, 60% 75% 80% 90% 95% 98% or 99% relative to the number of differentiated cells found in the first culture. Alternatively, "substantially continuously" can mean that the method is conducted for sufficient period of time that at least one, or at least two or at least three times the volume of media containing the first population of stem cells passes through the microfluidic device (a portion of which is returned to the first population of cells). "Substantially continuously" may also mean running the method, for 1 or more, 2 or more, 3 or more, or 4 or more, 8 or more, 12 or more, or 24 or more hours.

In another embodiment, the ordered array of obstacles comprises obstacles arranged in rows, wherein each subsequent row of obstacles is shifted laterally with respect to the previous row. Alternatively, the ordered array of obstacles may comprise obstacles arranged in rows, wherein each subsequent row of obstacles is shifted laterally with respect to the previous row or the ordered array of obstacles may be tilted at an offset angle e with respect to the direction of the field.

Microfluidic devices may also include a second array of obstacles within the microfluidic channel. This second array is fluidically coupled to, and downstream from, the first array, and comprises a network of gaps. A field propels the cells being separated through the first and second arrays, wherein a flux of the field from the gaps of the second array is divided unequally into a major flux component and a minor flux component that continues on to subsequent gaps in the network such that the average direction of the major flux component is not parallel to the average direction of the field. When introduced into the second array, cells having a size less than a critical size of the second array are transported generally in the average direction of the field, and cells having a size at least that of the critical size of the second array (second critical size) are transported generally in the average direction of the major flux component, thereby separating the cells. In such an embodiment, the stem cells may have either a critical size greater than the first critical size and less than the second critical size, or a critical size less than the first critical size and greater than the second critical size. The field responsible for propelling cells may be selected from fluid flow, centrifugal, gravitational, hydrodynamic, pressure gradient, or capillary action.

Microfluidic devices may also have one or more regions or obstacles that have a substance preferentially binding to one type of cell, a magnetic agent bound to the surface of one type of cell, or non-magnetic agent bound to the surface of one type of cell. In such embodiments, the substance may be selected from the group consisting of: charged organic polymers, uncharged organic polymers, nucleic acids, antibodies, avidin, biotin, carbohydrates, lectins, protein A, proteins and polypeptides. In cases where the substance is an antibody it may be an scFv, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv, or a disulfide linked Fv.

The microfluidic device may include a region for magnetic separation. In these cases, the methods may further comprise contacting a population cells with a magnetic agent, wherein the magnetic agent binds to one type cell and at least a portion of the population of the one type of cell is retained at the region for magnetic separation until release of the magnetic field. The magnetic agent may be of any suitable form known in the art. In one embodiment the magnetic agent is comprised of magnetic particles (e.g., silanized magnet particle) coupled to antibodies (see e.g., Decun, et al, J. Radio analytic, and Nuc. Chemistry, 206(2):l%9-200 (1996)). The antibodies may be specific to cell surface antigens, such as antigens on the surface of a stem cell to be isolated, or to antigens on the surface of a cell to be removed from a culture of stem cells (e.g., a terminally differentiated cell). Magnetic agents may be brought into contact with cells after their separation by size by introducing them into the microfluidic device through a separate input that combines the agent with only cells of one size.

In any of the foregoing embodiments or methods, the invention may further comprise contacting a population of stem cells with a non-magnetic agent that binds preferentially to one type of cell. The nonmagnetic agent can be retained or bound to a region of the device or to an obstacle within the device and bind to specific cells passing through. Thus, the cells may be retained at a surface of the device either temporarily or until eluted in response to a change in the surrounding environment. For example, the cells may be retained by ionic interactions and eluted in response to a change in salt concentration.

Non-magnetic agents that can bind to the surface of one type of cell include nucleic acids, antibodies, carbohydrates, lectins, proteins (e.g., antibodies) and polypeptides. In cases where the substance is an antibody, it may be an scFv, a Fab fragment, a Fab' fragment, a F(ab')₂, an Fv, or a disulfide linked Fv. Antibodies may be labeled with an agent such as biotin and then be bound to cells. These cells may then be retained at a surface in the device using avidin. Antibodies recognizing a cell surface antigen specific to a particular cell type may also be used directly to affinity purify cells by immobilizing the antibodies at one or more sites in a device, binding cells passing through the device and then releasing the cells in response to an elution protocol.

In some embodiments employing a magnetic or non-magnetic agent, the agent selectively binds to a compound specific for stem cells at a desired stage of differentiation (e.g., an antigen found on a multipotent, pluripotent, embryonic or adult stem cell) and is used to select those cells. In other embodiments, the agent may selectively bind to a compound found on the surface of a cell that must be removed from a population of cells. Thus, agents may be used to positively or negatively select cells. Among specific cells that may be targeted are totipotent, pluripotent, multipotent, oligopotent, or unipotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, neural crest stem cells, somatic stems cells, adult stem cells, or embryonic stem cells.

Antigens that may be targeted by antibodies include: CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD 105 CD349, CD 140b, CD324 and/or the mesenchymal/hematopoietic markers: CD105, CD90, CD73, CD44, CD29, HLA-A,B,C,

CD13, CD166, CD4ge, CD2711ow, CDlO, CD14, CD34, CD45, CDl 17, CD133, HLA-DR and/or the embryonic stem cell marker: SSEA-4. See e.g., Parolini, et al, "Concise Review:

Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells," Stem Cells 26:300-311 (2008).

In some embodiments, the array of obstacles will be an ordered array in which a principle axis of the array is not parallel to the direction of the field (FIG. 2). The term "asymmetric" refers to the array of obstacles (for example, to the array axis) and not to the shape of individual obstacles. In such an embodiment the array can be an ordered array so as to maximize the number of bifurcation events that can occur as the sample passes through the array (for example, see FIG. 3). In another embodiment, cells flow through an asymmetric obstacle array, and are separated according to size into different streams (FIG. 4). While smaller cells follow the field direction, the larger cells migrate in the array direction. The array direction corresponds to the average direction of the major component of the field flux (for example, see the gray arrow of FIG. 3). In instances where an obstacle array of the microfluidic devices employed in the above-described methods and the apparatus described below is asymmetrically aligned to the field, i.e., the obstacle lattice is asymmetric with respect to the average field direction, field lines going through one gap have to go around the obstacle in the next row. There exists a critical particle radius Ro above which particles move in the array direction (displacement mode), and smaller than which particles follow the average field direction (zigzag mode) (FIG. 8). By adjusting the array such that the critical size falls between the size of cells to be separated it is possible to separate populations of cells with a size greater than the critical size of one array and less than the critical size of the other array. Thus, where two or more arrays with different critical sizes are employed in a device it is possible to segregate three or more different populations of cells of different sizes by sorting cells passing from a first array on a second array (see FIGS. 9 andlO).

In some embodiments, the microfluidic devices employed may comprise an ordered array of obstacles and may employ a non-uniform field, wherein the direction of the field changes across the array. For example, changing field directions may be created by a curved microfluidic channel. Since the critical size of the array depends on the field direction, the non-uniform field creates sections of array with different critical sizes. This may be desired for fractionation of cells in a broad size range. In the particular embodiment shown in FIG. 13, the field direction is large at the first array section and then reduced at the following array sections, creating a large critical size in the first section and smaller critical sizes at the following sections.

In another embodiment, the microfluidic devices comprise an ordered square array of obstacles in a curved microfluidic channel (FIG. 14). The change in the field direction across the array creates a gradient of critical size in the array. Further, the field strength, another parameter that can be tuned, may be adjusted by changing the width of the microfluidic channel. This may result in a device with a better separation range and resolution than an array of one fixed critical size. A continuous gradient of critical size across the array can also be created by changing gap widths. Separations may also be improved using obstacles having different shapes or dimensions. The microfluidic devices employed in the methods and apparatus described herein may be prepared by any method known in the art, including those described in U.S. 7,150,812. The devices may be fabricated from materials that are compatible with the conditions present in the particular application of interest. Such conditions include, but are not limited to, pH, temperature, application of organic solvents, ionic strength, pressure, application of electric fields, surface charge, sticking properties, surface treatment, surface functionalization, bio-compatibility and conditions used for sterilization. The materials of the device may also be chosen for their optical properties, mechanical properties, and for their inertness to components of the application to be carried out in the device. Materials for microfluidic device construction include, but are not limited to, glass, fused silica, silicone rubber, silicon, ceramics, and polymeric substrates, e.g. plastics.

The microfluidic devices may have one or more portions (e.g., microfluidic channels) treated with a coating that prevents non-specific adhesion of cells to the device. To reduce non-specific adsorption of cells or compounds released by lysed cells onto the device, some or all portions may be coated with a thin film coating (e.g., a monolayer) of commercial non-stick reagents, such as those used to form hydrogels. Additional examples of chemical species that may be used to reduce non-specific adhesion include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose. A region of the device may be separately treated with materials that reduce nonspecific adhesion either before or after the device is assembled.

C. Apparatus The invention is also directed to devices for the culture or propagation of cells which include a chamber for maintaining cells in suspension (i.e., a state where they do not adhere tightly to the walls of the chamber and can be introduced into a microfluidic device substantially as a population of single cells). This chamber may be combined with any of the devices that are described above. For example the chamber may be in fluid connection with a microfluidic device having an inlet for receiving cells from the chamber and which separates cells based on size and/or affinity. The device may further comprise a first outlet for receiving cells having a size less than the first critical size and a second outlet for receiving cells having a size at least that of the first critical size. Either the first outlet or the second outlet may be in fluid connection with the chamber, thereby permitting cells to be returned to the chamber. Size separations in the devices may be accomplished using any of the means described for the devices above, including gaps, obstacles, pores and ordered arrays.

In some embodiments, less than 50%, 70%, 90%, 95%, 98%, or 99% of the cells having a size at least that of the first critical size introduced into microfluidic device are returned to the chamber for maintaining cells in suspension. In other embodiments less than

50%, 70%, 90%, 95%, 98%, or 99% of the cells having a size less than the first critical size are returned to the chamber.

Apparatus for the culture or propagation of cells may have a microfluidic device with both a first array and a second array with gaps, pores or spaces that separate cells of different sizes. The device may have a first outlet for receiving cells from the first array having a size greater than a first critical size, a second outlet for receiving cells from said second array having a size less than a second critical size and a third outlet for receiving cells from said second array having a size at least that of the second critical size. The devices may be designed so that the first critical size is less than the second critical size, and the second array receives a flow of cells from the first array having size at least that of the first critical size. Alternatively, devices may be designed so that the first critical size is greater than the second critical size, and so that the second array receives a flow of cells from the first array having a size less than the first critical size. The first outlet, second outlet, or third outlet of such devices may be in fluid connection with the chamber for maintaining cells in suspension.

Devices for the culture or propagation of cells may further comprise one or more regions or obstacles with a substance that preferentially binds to one type of cell, a magnetic agent bound to the surface of said one type of cell, or non-magnetic agent bound to the surface of said one type of cell. In some embodiments, at least one of the regions or obstacles is located between the inlet and the first array. In other embodiments, at least one of the regions or obstacles is located between the first array and the first outlet, between the first array and a second array, between the second array and a second outlet, or between the second array and a third outlet. The apparatus may have one or more regions or obstacles that comprise a substance that binds to cells and that is selected from the group consisting of charged organic polymers, uncharged organic polymers, nucleic acids, antibodies, avidin, biotin, carbohydrates, lectins, protein A, proteins and polypeptides. When the substance is an antibody, it may be an scFv, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv, or a disulfide linked Fv. These antibodies may be directed against an antigen selected from the group consisting of: CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD105, CD349, CD140b, CD324 and/or the mesenchymallhematopoietic markers: CD 105, CD90, CD73, CD44, CD29, HLA-A,B,C, CD13, CD166, CD49e, CD2711ow, CDlO, CD14, CD34, CD45, CDl 17, CD133, HLA-DR and/or the embryonic stem cell marker: SSEA-4 (see e.g., Parolini, et al., "Concise Review: Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells," Stem Cells 26:300-311 (2008)).

D. Isolation of Cells in Different Phases of the Cell Cycle

Cells vary in their size/volume over the phases of the cell cycle (see e.g., Rubin, et al., J. Applied Phys. (57(^:1585-1590 (1989)) and these variations can be exploited in isolation procedures. In order to obtain cells at a particular phase of the cell cycle, the cultures from which the cells are being isolated may advantageously be synchronized by halting the cell cycle at the desired phase. For example, serum starvation and treatment with thymidine or aphidicolin halts cells in the Gl phase; treatment with colchicine, vinblastin, or nocodazole halts cells in the M phase; and treatment with 5- fluorodeoxyuridine halts cells in the S phase. Cells in different phases of the cell cycle can then be isolated by sorting a population of cells, especially a clonal population of cells, using any of the devices for size separation described herein. This includes devices with, or without chamber for maintaining cells in suspension and with microchannels having gaps, obstacles pores, etc. As discussed previously, devices may have one or more arrays, one or more outlets and may, or may not, include substances such as antibodies that bind to cells to aid in separations. Size selection may be done in the presence of agents (e.g., cholcine) or treatment (e.g., serum starvation) that induce the synchronization of the culture alone or in combination with a reduced temperature for the separation process. When reduced temperatures are used the apparatus must be suitably temperature controlled. In some embodiments the microfluidic device is configured such that the mitotic cells (or cells at another desired stage of the cell cycle) have a size that is greater than the critical size of a first array and less than the critical size of a second array. In other embodiments the microfluidic device is configured such that cells having of a size corresponding to that of a cell undergoing mitosis have either a critical size greater than a first critical size and less than a second critical size, or a critical size less than a first critical size and greater than a second critical size.

All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

What is Claimed is:

1. A method of treating a first population of stem cells in order to produce a second population that has been enriched in stem cells at a specific state of differentiation, said method comprising: a) introducing a first population of stem cells into one or more micro fluidic channels of a micro fluidic device, wherein: i) said first population of stem cells is in the form of a suspension of cultured cells comprising: stem cells in a first differentiation state (A), wherein said cells have an average diameter x; and stem cells in a second differentiation state (B), wherein said cells have an average diameter y that is different from x and are in the same lineage as A; ii) said microfluidic channels comprise gaps, pores or obstacles that have a diameter effective for separating cells of diameter x from cells of diameter y; b) separating said A stem cells from said B stem cells by propelling said first population of stem cells thorough said one or more microfluidic channels to one or more outlets on said microfluidic device; and c) collecting said second population of stem cells from said one or more outlets wherein, relative to said first population, said second population has been enriched in cells in either said first or said second differentiation state.

2. The method of claim 1, wherein said stem cells in said first differentiation state (A) are totipotent stem cells and said stem cells in a second differentiation state (B) are pluripotent, multipotent, oligopotent or unipotent stem cells.

3. The method of claim 1, wherein said stem cells in a first differentiation state (A) are pluripotent stem cells and said stem cells in a second differentiation state (B) are multipotent, oligopotent or unipotent stem cells.

4. The method of claim 1, wherein said stem cells in said first differentiation state (A) are multipotent stem cells and said stem cells in a second differentiation state (B) are oligopotent or unipotent stem cells.

5. The method of claim 1, wherein said stem cells in said first differentiation state (A) are oligopotent stem cells and said stem cells in a second differentiation state (B) are unipotent stem cells.

6. The method of claim 1, wherein said second population has been enriched in totipotent stem cells.

7. The method of claim 1 , wherein: a) said microfluidic device comprises a first array, said first array comprising a network of gaps within said one or more microfluidic channels; b) said stem cells are propelled through said one or more microfluidic channels by a field and a flux of the field from the gaps is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field; c) cells having a size less than a predetermined first critical size between x and y are transported generally in the average direction of the field, and cells having a size at least that of the first critical size are transported generally in the average direction of the major flux component, thereby separating the cells according to size.

8. The method of claim 1 , wherein: a) said microfluidic device comprises an ordered first array of obstacles within said one or more microfluidic channels; b) wherein the device employs a field that propels the cells being separated through the microfluidic channel; c) said ordered first array of obstacles is asymmetric with respect to the average direction of the field, such that, when cells are introduced into the first array, cells having a size less than a predetermined first critical size between x and y are transported in a first direction, and cells having a size at least that of the first critical size are transported in a second direction, wherein said first and second directions are different, thereby separating the cells according to size.

9. The method of either claims 7 or 8, wherein said microfluidic device further comprises a second array of obstacles within said one or more microfluidic channels, and wherein: a) said second array is fluidically coupled to, and downstream from, said first array; b) said second array comprises a network of gaps within said one or more microfluidic channels; b) said stem cells are propelled through said first array and said second array by a field and a flux of the field from the gaps of the second array is divided unequally into a major flux component and a minor flux component such that the average direction of the major flux component is not parallel to the average direction of the field, and, when cells are introduced into the second array, cells having a size less than a predetermined critical size of said second array are transported generally in the average direction of the field, and cells having a size at least that of the critical size of the second array are transported generally in the average direction of the major flux component, thereby separating the cells according to size.

10. The method of any one of claims 7-9, wherein said field is due to fluid flow, or due to centrifugal, gravitational, hydrodynamic, pressure gradient, or capillary action.

11. The method of any one of claims 1-10, wherein said microfluidic device further comprises one or more regions or obstacles comprising a substance that preferentially binds to: one type of cell; a magnetic agent bound to the surface of one type of cell; or a non-magnetic agent bound to the surface of one type of cell.

12. The method of claim 11, wherein said substance selectively binds to a protein or antigen found on the surface of an embryonic stem cell or an adult stem cell as compared to a differentiated cell arising from said embryonic stem cell or an adult stem cell.

13. The method of claim 12, wherein said substance is selected from the group consisting of: a monospecific polyclonal antibody, monoclonal antibody, scFv, Fab fragment, a Fab' fragment, a F(ab')₂, Fv, or a disulfide linked Fv directed against antigen.

14. The method of claim 13, wherein said antigen is selected from the group consisting of: CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD105 CD349, CD140b, CD324; CD44, CD29, HLA-A,B,C, CD13, CD166, CD49e, CD2711ow, CDlO, CD14, CDl 17, CD133, HLA-DR and SSEA-4.

15. The method of any one of claims 1-14, wherein the second population of stem cells collected in step c), is tested for a cell surface stem cell marker.

16. The method of claim 16, wherein said cell surface stem cell marker is selected from the group consisting of: CD34, CD45, CD36, GPA, CD71, CD73, CD90, CD105 CD349, CD140b, CD324; CD44, CD29, HLA-A,B,C, CD13, CD166, CD49e, CD2711ow, CDlO, CD14, CDl 17, CD133, HLA-DR and SSEA-4.

17. An apparatus for the culture or propagation of cells comprising:

(a) a chamber for maintaining cells in suspension;

(b) a micro fluidic device comprising: i) an inlet for receiving said cells in suspension from said chamber, wherein said inlet is in fluid connection with said chamber; ii) a microfluidic channel comprising a first array, wherein said first array comprises a network of gaps within the microfluidic channel; iii) a first outlet for receiving cells from said first array wherein said cells have a size less than a first critical size and a second outlet for receiving cells from said first array wherein said cells have a size at least that of the first critical size.

18. The apparatus of claim 17, wherein said first outlet is in fluid connection with said chamber thereby permitting cells having a size less than said first critical size to be returned to said chamber.

19. The apparatus of claim 17, wherein the second outlet is in fluid connection with said chamber thereby permitting cells having a size at least that of the first critical size first critical size to be returned to said chamber.