Patent Application
20120202284
Existing methods of cell identification and separation are problematic and often unsatisfactory. For example, difficulties experienced in detection and sorting of stem cells are a significant barrier to the use of stem cells for the manufacturing of replacement organs.
Current sorting techniques such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) require that cells are modified by the attachment of a suitable marker for subsequent detection.
Such techniques can suffer from slow sorting speeds, low cell yields and cell damage.
By way of non-limiting examples, embodiments are now disclosed. In a first embodiment, apparatus for sorting cells is provided, including a cell delivery device, a reflection surface, a first collection area and a second collection area. In this first embodiment, the delivery device is configured to deliver two or more cells along a flow path, the two or more cells including a first type of cells having a first mechanical stiffness and at least a second type of cells having a second mechanical stiffness different from the first mechanical stiffness. The reflection surface is provided across the flow path at an oblique angle relative to the flow path and configured to reflect the cells at reflection angles relative to the flow path. In this first embodiment, the first collection area is positioned to receive the first type of cells reflected from the reflection surface substantially at a first reflection angle; and the second collection area is positioned to receive the second type of cells reflected from the reflection surface substantially at a second reflection angle different from the first reflection angle. The difference in the first and second reflection angles is dependent on the mechanical stiffness of the first and second types of cells.
In a second embodiment, a method of sorting cells is disclosed. The method includes causing two or more cells travelling along a flow path to contact a reflection surface and to rebound at different reflection angles, the cells including a first type of cells having a first mechanical stiffness and at least a second type of cells having a second mechanical stiffness different from the first mechanical stiffness; collecting the first type of cells reflected from the reflection surface substantially at a first reflection angle; and collecting the second type of cells reflected from the reflection surface substantially at a second reflection angle different from the first reflection angle. The first and second reflection angles are different dependent on the mechanical stiffness properties of the first and second types of cells.
In a third embodiment, another apparatus for sorting cells is disclosed. The apparatus in the third embodiment includes means for producing a flow of two or more cells, impact means, upon which the cells are impactable, wherein impact with said impact means causes the cells to travel to different locations relative to the impact means as a result of differences in kinetic energy loss of the cells upon impact; and means for collecting the cells at the different positions.
In this example, the cells 110, 120 include at least two types of cells, a first type of cells 110 having substantially a first mechanical stiffness and at least a second type of cells 120 having substantially a second mechanical stiffness different from the first mechanical stiffness.
The apparatus 100 is configured to sort at least the first type of cells 110 from the second type of cells 120 partially or completely in view of the different mechanical stiffness of the different cell types. Sorting may include the separation or partial separation of the at least first and second types of cells, the cells being separated, as a result of the sorting, into two or more different cell samples. The separation may be partial in the sense that, even after sorting, there may be cells of both the first and second types of cells in one or more of the cell samples. In this example, the second mechanical stiffness is lower than the first mechanical stiffness. By delivering the cells 110, 120 towards the surface 140, the cells can impact (i.e., collide) with the surface 140 and reflect from the surface 140 at an angle relative to the incident flow path 151. At least due to the difference in mechanical stiffness, the second type of cells 120 in this example reflect at a greater reflection angle relative to the normal of the reflection surface 140 than the first type of cells 110. Thus, at least the first and second types of cells 110, 120 can travel along diverging reflection flow paths, after reflecting from the surface 140, the reflection flow paths indicated generally by second and third arrows 152, 153 in
It is understood that there may be variations in the stiffness of cells within a population of a single type of cells. In general, the differences in stiffness between the different types of cells to be sorted will be greater than the differences in stiffness in a pure population of at least one of the different types of cells. As an example, with reference to Pajerowski, J. D., et al., Physical plasticity of the nucleus in stem cell differentiation, PNAS (2007) Oct. 2, vol. 104, no. 40, 15619-15624, FIG. 1, variation in stiffness of differentiated and undifferentiated stem cells, in respective populations of these types of cells, may be about +/−50% of the average stiffness in the populations. This variation may be partly due to the variation in properties of cells and partly due to measurement error. Thus, for undifferentiated and differentiated stem cell populations, each population exhibiting a variation in average stiffness, efficient sorting may be achieved where the difference between the average stiffness of the two different populations is at least about 100%. With reference to Tan S. C. et al., Viscoelastic behaviour of human mesenchymal stem cells, BMC Cell Biol. 2008 Jul. 22; 9:40, and Pajerowski, J. D., et al, the elastic modulus of stem cells may range from approximately 900 Pa for undifferentiated cells to 5400 Pa for fully differentiated cells. Since this gives an approximately 600% (6-fold) difference in stiffness between these two types of cells, any +/−50% variation of the average stiffness in respective populations of each of these two types of cells may not be significant. Thus, efficient sorting of undifferentiated and differentiated stem cells is described as one example of types of cells that may be sorted in apparatus and methods described herein.
The reflection surface may be a rigid surface or a non-rigid surface. When a rigid surface is used there may be substantially no flexing or movement of the surface upon impact with the cells. The surface may be sufficiently rigid to minimise absorption of energy from the cells. Any flexibility may result in loss of rebound velocity and therefore diminish the yield. Examples of materials that may have sufficient rigidity include thermoplastics and stainless steel. The reflection surface may be entirely coated, partially coated or uncoated. For example, a coating may be applied at least partially to the reflection surface to alter friction properties of the surface. For example, the coating may be provided to increase the coefficient of friction of the surface, to reduce or prevent slippage of the cells upon impact with the surface, or to decrease the coefficient of friction, to reduce or prevent sticking of the cells upon impact with the surface. The coefficient of friction of the reflection surface may additionally or alternatively be altered by providing a surface with a particular roughness, with or without a coating applied to the surface. By reducing or preventing sliding or sticking of the cells, the possibility of cells reflecting in an unpredictable manner may be reduced or prevented, making it more straightforward to sort the cells. As another example, the reflection surface may additionally or alternatively be coated with a hydrophobic and/or hydrophilic coating to reduce or prevent sticking or slipping of the cells. On the whole, the surface may be configured, whether coated or not, to minimise slippage and/or sticking of cells and to provide consistent uniformity and rigidity properties for different types of cells that are to impact with the surface. Coatings that may be suitable include but are not limited to Teflon™ (polytetrafluoroethylene (PTFE)) or other fluorinated polymers, which, in addition to their friction properties, may have desired hydrophobicity properties. Polymers such as but not limited to 2-hydroxyethyl methacrylate (HEMA) and other methacrylates may be used to increase hydrophilicity Roughened surfaces may include physically post-treated polymer surfaces, such as physically abraided polyethylene, or a patterned surface created by micromachining ion milled film, for example.
Prior to delivery from the nozzle, the cells may be combined with a column of pressurized sheath fluid. A piezo transducer may be used to break up the suspension of cells in the fluid into a rapidly moving stream of droplets, ejected from the nozzle. The nozzle may be a conventional nozzle used in FACS equipment, or otherwise. In an illustrative embodiment, a FACS nozzle arrangement which may be used in the present apparatus is described in Derek Davies, Chapter 5: Cell Sorting by Flow Cytometry in Flow Cytometry: Principles and Applications, edited by M. G. Macey, Humana Press Inc., Totwa N.J. The flow rate of cells will depend on the sheath pressure, the nozzle size, and the number of cells. However, as an example, with a nozzle size of 50 microns and a sheath pressure of 80 psi, 160,000 drops per second may be delivered from the nozzle. If there is, for example, 1 cell in every four drops, 40,000 cells per second may therefore be delivered from the nozzle, for example. The velocity of the cells will also vary depending on the sheath pressure, but may be a velocity of approximately 10 m/s for a sheath pressure of 12 psi and approximately 50m/s for sheath pressure of 80 psi. In general, however, many different approaches may be taken to delivering a flow of cells to the reflection surface.
One or more separation elements, particularly a wedge 160 in this example, can be employed to maintain separation of cells 110, 120 as they travel at different reflection angles along the different reflection flow paths 152, 153 after reflecting from the reflection surface 140. The one or more separation elements may define two or more collecting areas for the reflected cells. The wedge 160 in this example has a wedge angle corresponding substantially to the difference in angle of the reflection angles of the first and second cell types 110, 120, although other wedge angles may be employed, e.g., to adjust a degree of divergence between the flow paths 152, 153. To change the wedge angle, the wedge 160 may be interchangeable with other wedges or the wedge itself may be adjustable or moveable. The wedge, or another type of separation element, may be adjusted or moved in accordance with the angle of difference between the reflection angles of the first and second cell types (e.g., so that the wedge angle substantially matches the angle of difference), or to adjust the degree of divergence between the flow paths of the reflected cells as desired. As one example, the wedge may be adjustable by comprising two opposing walls, angled relative to each other, the walls converging to an apex of the wedge shape, wherein the walls are pivotably connected at the apex, optionally by a hinge. A mechanism, such as a screw mechanism or linear actuator, for example, located between the two opposing walls, may be operable to relatively pivot the walls, although other mechanisms may be employed.
One or more separation elements other than a wedge may be used in the example illustrated in
The orientation and/or distance of the separation element from the reflection surface may be chosen depending on one or more factors such as but not limited to the incident angle of the cells on the reflection surface, the width of the stream of cells incident on the reflection surface (the ‘beam width’), the difference in the reflection angles between the different cell types reflecting from the reflection surface, the velocity of the reflected cells, and the effects of possible external factors on the cells after reflection, such as gravity, air currents, or other external forces that might act on the cells to change their speed and/or direction of motion. As an example, when choosing where to position one or more separation elements, relative to one or more reflection surfaces, the minimum vertical distance, dv, from the central point of impact on the reflection surface of a stream of incident differentiated and undifferentiated cells may be calculated using the Formula dv=b(√Cd.√Cu/(√Cd−√Cu))/sin θ, where dv is the vertical distance from centre plane of impact; θi is the incident angle, Cd and Cu are the coefficient of restitution of differentiated and undifferentiated cells, respectively, and b is the beam width. Thus, for a beam width of 50 μm, incident angle of 60 degrees, Cd and Cu of 0.8 and 0.7 respectively, dv is approximately 1 mm and therefore the separation between the reflection surface and the separation element may be at least 1 mm. In practice, however, the separation may be greater than this minimum difference, e.g. double the minimum distance or otherwise.
Although only one reflection surface 140 is shown in
As indicated, it is recognised that one or more cells of the first type of cells may still be interspersed with one or more cells of the second type of cells in cell samples, even after a first separation. In these circumstances, the separated cell samples may be considered to not be completely ‘pure’. To increase purity, one or more separated cell samples may be recirculated through the apparatus by being fed back through the nozzle 130 and subjected to the same sorting process again, or by being fed through the nozzle 130 and subjected to the sorting process with one or more variables changed, such as, but not limited to, the angle of the reflection surface relative to the incident angle, or the nozzle speed, etc. The recirculation process may be a closed loop process, where the same collection of cells is recirculated only, or an open-loop process, where additional cells to be sorted are introduced into the system at the same time as cells are being recirculated through the sorting process.
Additionally or alternatively, one or more of the separated cell samples may be subject to one or more additional sorting stages ‘upstream’ or ‘downstream’, (i.e. before or after, respectively, the sorting as described above with respect to examples herein), which additional sorting stages may employ one or more additional reflection surfaces and/or separation elements and may operate under the same sorting principles already discussed, or may employ different sorting elements, apparatus and/or sorting principles, etc. For example, a separated cell sample may be subjected to cell cytometry sorting apparatus ‘downstream’. As another example, a rough cell sorting for viable cells may be carried out ‘upstream’, which cells are then fed into apparatus as described herein.
The application of the cells to a recirculation process or to further sorting stages may be exercised automatically in certain embodiments. For example, detection apparatus may be provided to automatically detect the purity of separate cell samples, determine whether the purity meets a desired level of purity, e.g. the total number of cells includes 70% or more, 80% or more, 90% or more, or 95% or more, of one cell type only, and, on this basis, determine whether or not to subject the cells to further sorting processes. Detection of the purity of cell samples may be performed taglessly, by visual imaging and identification of cells by morphology. Alternatively, detection may be performed with tags, for example, by cell-type specific molecular tags of a fluorescent, and/or magnetic type.
It should be understood that the techniques described herein may be automated using a variety of technologies. For example, one or more of the steps described herein may be initiated, or cell sorting parameters may be adjusted, using a series of computer executable instructions residing on a suitable computer readable medium. For example, computer executable instructions may control one or more switching elements that may optionally be included in the apparatus, such as a switching element configured to turn the delivery of cells from the nozzle ‘on’ or ‘off’. As another example, computer executable instructions may control one or more motorized elements, e.g. one or more linear actuators or piezo-electric motors, that may optionally be included in the apparatus, to relatively pivot walls of a wedge element or relatively pivot the nozzle and the reflection surface to allow the incident angle to be varied, etc. Suitable computer readable media may include volatile (e.g. RAM) and/or non-volatile (e.g. ROM, disk) memory, carrier waves and transmission media (e.g. copper wire, coaxial cable, fibre optic media). Exemplary carrier waves may take the form of electrical, electromagnetic or optical signals conveying digital data streams along a local network or a publically accessible network such as the Internet
Additionally or alternatively, to increase purity, the one or more separation elements, e.g. the wedge, may be positioned or modified in a biased manner, so that they separate, at least on one side, cells that have a significantly different reflection angle to certain other reflected cells. By having a significantly different reflection angle to certain other reflected cells, those cells may be more likely to have a significantly different stiffness to the other reflected cells, making it more likely that they are of a particular cell type, for example. This approach may ensure that at least a cell sample separated to one side of the separation element has a desired level of purity, although this may be at the expense of the yield (i.e. total number of cells) of that separated sample, and the purity of one or more other separated cell samples.
In general, the desired level of purity of separated cell samples may depend on the application or end use of the separated cell samples (e.g., therapeutic use may require a greater purity than research use).
Although only two types of cells having different mechanical stiffness are shown in the example illustrated in
In another approach, more than two collection areas may be provided to collect a respective one of the more than two different types of cells, after reflection from the one or more reflection surfaces. In these circumstances or otherwise, a plurality of separation elements may be used to maintain separation of the cells. For example, if there are three types of cells to be sorted, at least two separation elements may be provided to maintain three types of separated cells apart after reflection from the one or more reflection surfaces, at least one of the separation elements being located directly between two of the three cell types and at least one other of the separation elements being located between a different two of the three cell types.
In some circumstances, the different cell types present in a sample may be known prior to sorting, e.g., through fluorescent detection or visual imaging for cell morphology, and sorting may be performed to separate one cell type from one or more other cell types, and/or each cell type from the other. In other circumstances, the different cell types present in the sample may be completely or partially unknown prior to sorting and the apparatus may be used to separate the different types present as well as to optionally identify different types of cells through the sorting process, based on where the cells are collected, for example. Thus, in some circumstances, it may be assumed that, due to a particular cell being collected in a particular collection area, that cell may have particular mechanical stiffness properties, which particular stiffness properties may be indicative of its cell type.
In the example illustrated in
As indicated, the cells 110, 120 reflect at different reflection angles dependent on differences in mechanical stiffness. Mechanical stiffness is indicative of the resistance offered by a body to deformation. The stiffer a cell, the less it will deform upon collision with a surface, e.g., in accordance with principles of a simple mechanical spring. Since collision and deformation are, in practice, somewhat inelastic, a greater degree of deformation can correspond to a greater kinetic energy (KE) loss by a cell on collision. As a result of the differences in deformation and kinetic energy loss, cells can behave differently when they reflect from a surface. Cells with greater relative stiffness can have a greater velocity when they reflect from a surface (i.e., a greater reflection velocity) than cells with lower relative stiffness. When the cells collide with a surface at an oblique angle relative to their flow path, cells with greater relative stiffness can reflect from the surface at a smaller reflection angle to the normal of the surface (e.g., an angle closer to the incident angle relative to the normal of the surface) than cells with lower relative stiffness. By travelling at different reflection velocities and/or at different reflection angles, cells with different mechanical stiffness can travel to different positions in free space, as indicated in
The apparatus 100 may be used to sort a variety of different types of cells, including human, mammalian or animal cells. It is not intended that apparatus or method disclosed herein be necessarily limited to sorting any particular cell type.
For example, the apparatus or method may be used to sort stem cells. Stem cells include cells that have the capacity to self renew (i.e., go through cycles of cell division while maintaining the undifferentiated state) and to differentiate into specialized cell types. Stem cells can be either totipotent or pluripotent, although multipotent, oligopotent, or unipotent progenitor cells may also be considered as stem cells. Differentiation of stem cells may increase mechanical stiffness of cells. For example, undifferentiated embryonic stem cells exhibit greater deformability (up to six times greater, for example) than fully differentiated stem cells. Cell stiffness properties of differentiated stem cells are discussed in Pajerowski, J. D., et al., Physical plasticity of the nucleus in stem cell differentiation, PNAS (2007) Oct. 2, vol. 104, no. 40, 15619-15624, for example.
Thus, the apparatus or method may be used to sort cells that have different degrees of differentiation. For example, non-differentiated stem cells may be sorted from differentiated stem cells. As another example, differentiated stem cells may be sorted from progressively more differentiated stem cells. The apparatus may also be used to sort non-differentiated or partially differentiated stem cells from terminally differentiated cells.
As an example, undifferentiated embryonic stem cells may be sorted from further differentiated counterparts. As another example, adult stem cells with stiffness characteristics varying depending on their stem cell type and degree of differentiation may be sorted.
In addition to stem cells, the apparatus or method may be used to sort other types of cells which vary in stiffness. For example, cancerous cells may have a different stiffness to their normal cell counterparts and therefore the apparatus and method may be used to separate such cells. Metastatic cancer cells, for example, are less stiff than regular mesothelial cells. By permitting sorting of such cells, the apparatus or method described herein may be used in a diagnostic technique.
In general, a cell type that is sorted from another cell type may itself include different kinds of cells. The different kinds of cells may have similar mechanical stiffness properties in comparison to the type of cells that they are to be sorted from. As one non-limiting example of this, one cell type might include a collection of different stem cells having different degrees of differentiation, and these stems cells may be sorted collectively from a cell type including terminally differentiated cells only. In general, a ‘cell type’ may include a broad range of cells that may have mechanical stiffness properties falling within in a predetermined range, for example.
Example mathematical analysis of behaviour of cells as they reflect from a surface follows. The analysis may be used to at least partially determine, for example, appropriate settings and configurations for the sorting apparatus or method, such as the incident velocity, incident angle and/or positioning and orientation of a reflection surface, one or more separation elements and/or collection devices for collecting cells, or otherwise. However, it is conceived that various approaches may be taken to analysing and predicting behaviour of cells, which may use different mathematical formulae to the formulae presented and/or use experimentation and observation, for example. Overall, understanding mathematical theory behind the behaviour of cells is not necessary to implement embodiments and examples disclosed herein.
A value for the work done Win deforming a cell upon impact with a surface can be determined using Formula 1:
W=−πE{p3[A−1/3 ln(p)]+p[r2 ln(p)−B]+C} Formula 1
where p=(r−d), A=1/3 ln(r)+1/9, B=r2 ln(r)+r2 and C=8/9r3, and where E is the modulus of elasticity of the cell, r is the radius of the cell and d is the total deformation of the cell.
Formula 1 assumes at least that cells are approximately spherical, the stiffness properties of the cells are homogeneous throughout each cell, the collision surface is flat, and there is no cell slippage upon collision with the surface.
Formula 1 may be used to determine an approximate value for the total deformation of a cell by substituting quantitative values for E, r, m and v using the relation:
W=KE=1/2mv2 Formula 2
where m is the cell mass and v is the velocity of the cell incident on the surface (incident velocity).
Formula 1 indicates that, at least when velocities and masses of cells are substantially the same, and thus their kinetic energies are also substantially the same, cells of higher relative stiffness (with higher Elastic modulus) will be deformed less than cells of lower relative stiffness (with lower Elastic modulus).
Assuming that, in practice, cell deformation is not energy conserving due to losses in the collision with the surface, larger relative deformation will result in larger relative kinetic energy loss for cells of lower relative stiffness, resulting in a relatively lower reflection velocity for such cells. With reference to
As indicated, a value for the deformation, d, for different cell types can be calculated using Formula 1. To calculate a reflection angle, θr, for cells, d can be inserted into Formula 3, which assumes that the loss of energy in the collision is proportional to the effective strain (effective strain being expressed as the ratio of the deformation over the cell diameter).
KE-reflected=[1−(d/cell diameter)]×KE-incident Formula 3
In Formula 3, KE-reflected and KE-incident describe the kinetic energy of the cells normal to the surface, after and before reflection. Referring to Formula 2, values for KE-incident can be determined from the cell mass and incident velocity (which may correspond substantially to the nozzle velocity of a delivery device), taking into consideration the incident angle of the cell on the reflection surface. By obtaining KE-reflected, again with reference to Formula 2, the reflection velocity of the cell normal to the surface, vrn, can be calculated and reflection angles can then be calculated using the following formula, Formula 4:
θr=tan−1(vrt/vrn) Formula 4
Taking non-differentiated and differentiated stem cells as a non-limiting example of cells that may be sorted, and using the following possible properties for stem cells, and example approximate values for incident velocities and incident angle, deformation, d, velocities, vrn, and reflection angles θr, can be calculated as follows:
The calculations indicate a difference in reflection angle for the differentiated and non-differentiated cells of about 3 degrees based on this example mathematical approach.
Nonetheless, in practice, cells are not perfectly elastic and they more closely match a viscoelastic model. An alternative measure of energy loss during impact may therefore be coefficient of restitution, which may range from about 0.5 for cells of higher relative stiffness to 0.1 for the cells of lower relative stiffness, for example. Taking this into account, a larger difference in reflection angle between different types of cells may be predicted. The difference may be 20 degrees, rather than 3 degrees, for example.
Furthermore, in practice, a population of cells may not be completely homogenous in terms of such physical properties as size, density and shape, and some variation is expected. This can affect the reflection velocities and angles.
Referring to Tables 1a and 1b, and in consideration of stem cell and apparatus parameters as discussed herein previously, a variation of 10% in cell radius can vary the difference in the reflection angles of undifferentiated and differentiated stem cells by about 3%, for example. Similarly, a variation of 20% in density can vary the difference in the reflection angles of undifferentiated and differentiated stem cells by about 10%, for example.
To address variability in cell population, one or more separated cell samples may be cycled through the same apparatus more than once, or may be subjected to one or more additional sorting stages ‘downstream’, which additional sorting stages may employ one or more additional reflection surfaces and separation elements and operate under the same sorting principles or otherwise. In general, subjecting cells to additional sorting processes may increase enrichment or sample purity and increase cell yields. As an alternative, or additionally, incident velocity or incident angle may be varied to tune the apparatus for particular populations of cell types.
As indicated, in addition to physical properties of cells to be sorted, the reflection velocities and reflection angles can be affected by the incident velocity and incident angle of the cells, as they collide with the reflection surface. The incident velocity may largely depend on the velocity of the cells as they leave the nozzle, i.e., the nozzle velocity. The incident velocity may be kept below a level at which little or no damage to cells will occur upon collision with the reflection surface, or with any elements of the apparatus further downstream. For example, the incident velocity of the cells may be of the same order of magnitude as used in existing FACS equipment as discussed above, to the extent that it does not damage cells. Nonetheless, cell damage may also be reduced or prevented by encasing the delivered cells in fluid droplets and this may permit greater incident velocities to be used. The droplets can include cells with sheath fluid such as water encasing them. The droplets may be located in air as they move towards and away from the reflection surface. The combination in a droplet of the fluid and a cell can have a different stiffness to the cell by itself. However, the relative stiffness of droplets containing cells of different stiffness, within the same type of fluid, can remain approximately the same. Although, using conventional FACS nozzle arrangements, cells will normally be encased in a sheath fluid at least during delivery to the one or more reflection surfaces, it is recognized that a flow of cells may produced by alternative means. It is considered that delivery of a flow of cells, which cells are not encased in fluid droplets, may utilized in the apparatus and methods described herein. The cells may be delivered in a gas medium only, such as air, for example. Alternatively, rather than being encased in respective fluid droplets, the cells may disposed in a larger fluid medium, as they travel to and/or from the reflection surface. As an example, the cells may be delivered to the reflection surface, and to the collection areas, via in one or more microfluidics channels containing fluid.
The incident velocity may be of an order of magnitude lower than stresses experienced by cells in various natural processes (e.g., cell division). The incident angle may be chosen to ensure a maximum difference in the reflection angle, dependent on the types of cells being sorted, for example. It is considered that an incident angle of between about 30 and 45 degrees, or 40 degrees and 50 degrees, e.g., 45 degrees, may be appropriate, but other angles may be used. Although the incident angle is fixed in
One or more of the conduits may have a cross-section perpendicular to their elongation direction that is square, rectangular, circular or otherwise. The internal walls of the conduits may be smooth and/or provided without sharp corners (e.g. acute angles) to minimize cells or droplets containing cells from getting stuck, attached, and/or damaged. To provide smooth corners, the corners may be rounded or filleted. Where rounded or filleted corners are provided, the radii of these corners may be greater than that of the cells or droplets to reduce chances of sticking The shapes and/or materials of the conduits may be chosen so that the conduits do not damage cells passing therethrough by either chemical reaction or by physical damage.
The size of the conduits may depend on the size or number of nozzles and/or the nature of the cells being sorted, for example. If a single 20 μm nozzle is used, the delivery conduit 220 may have a 40 μm×40 μm square cross-section and the reflection conduits may have a 30 μm×40 μm rectangular cross-section, for example. Multiple nozzles may be used in apparatus disclosed herein to increase the number of cells being sorted in a given time. The multiple nozzles may be provided side-by-side and may deliver cells through the same conduit(s) such that they are incident on the same reflection surfaces and collected in the same collection areas, etc., or the nozzles may deliver the cells through different respective conduits, such that they are incident on different respective reflection surfaces and collected in different collection containers, etc. When different conduits, etc., are provided, in essence, a plurality of apparatuses as illustrated in the Figures or otherwise may be used in parallel, to increase the number of cells sorted in a given time.
In this example shown in
In the examples provided, sorting of cells is dependent on the stiffness of different types of cells and therefore damage to cells may be significantly reduced or avoided in comparison to approaches in which a marker is applied to cells. Furthermore, the purity of sorted or separated cell samples may reach 70% or more, 80% or more, 90% or more or 95% or more. This may be achieved with a difference in reflection angle for different cell types of at least 2 degrees, 3 degrees, 5 degrees, or 10 degrees, for example. Purity may be increased by tuning of the apparatus or introducing multiple processing stages in the apparatus as discussed. The rate of cells being sorted may be higher in comparison to approaches where markers are applied to cells, since a marking stage may be eliminated and multiple nozzles delivering cells may be used.
It will be appreciated that numerous variations and/or modifications may be made to the examples. For instance, a variety of different configurations of elements of the apparatus described in the examples and as illustrated in the Figures, including the nozzles, housings, separation elements, reflection surfaces or collection containers, etc. is conceivable. Furthermore, such elements of the apparatus, described with respect to one example, may be interchangeable with one or more corresponding elements of one or more other examples, or may be provided as additional elements of one or more other examples. For example, collection containers similar or identical to the collection containers of the apparatus illustrated in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU11/00123 | 2/7/2011 | WO | 00 | 8/30/2011 |
