U.S. patent application number 14/079319 was filed with the patent office on 2016-12-29 for microstructure for particle and cell separation, identification, sorting, and manipulation.
The applicant listed for this patent is ANGLE North America, Inc.. Invention is credited to Georgi HVICHIA.
Application Number | 20160376553 14/079319 |
Document ID | / |
Family ID | 23184670 |
Filed Date | 2016-12-29 |
United States Patent
Application |
20160376553 |
Kind Code |
A9 |
HVICHIA; Georgi |
December 29, 2016 |
Microstructure for Particle and Cell Separation, Identification,
Sorting, and Manipulation
Abstract
The invention relates to microscale cell separating apparatus
which are able to separate cells on the basis of size of the cells,
interaction of the cells with surfaces of the apparatus, or both.
The apparatus comprises a stepped or sloped separation element (16)
interposed between an inlet region (20) and an outlet region (22)
of a void that can be tilled with fluid. The void can be enclosed
within a cover (12) and fluid flow through the void engages cells
with the separation element. Only cells which have (or can deform
to have) a characteristic dimension smaller than or equal to the
distance between a step and the cover or body can pass onto or past
a step. Modifications of surfaces within the apparatus can also
inhibit passage of cells onto or past a step.
Inventors: |
HVICHIA; Georgi;
(Philadelphia, PA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
ANGLE North America, Inc. |
Philadelphia |
PA |
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20140072952 A1 |
March 13, 2014 |
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Family ID: |
23184670 |
Appl. No.: |
14/079319 |
Filed: |
November 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13170369 |
Jun 28, 2011 |
8765456 |
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14079319 |
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10587053 |
Dec 11, 2006 |
7993908 |
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PCT/US02/22689 |
Jul 17, 2002 |
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13170369 |
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60306296 |
Jul 17, 2001 |
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Current U.S.
Class: |
435/2 ; 210/767;
210/808 |
Current CPC
Class: |
C12M 47/04 20130101;
C12N 5/0606 20130101; G01N 2015/0288 20130101; B01L 2400/0481
20130101; C12M 47/02 20130101; B01L 2200/10 20130101; B01L 2300/04
20130101; G01N 15/0272 20130101; B01D 35/30 20130101; B01L 3/502761
20130101; C12N 5/0603 20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; B01D 35/30 20060101 B01D035/30 |
Claims
1. A method of separating particles, the method comprising
providing the particles to the inlet region of a microscale
apparatus, the apparatus comprising a body, a cover, and a
separation element, the body and cover defining a void having an
inlet region, an outlet region, and a surface, the separation
element i) being disposed in the void, ii) having a plurality of
steps including a first step and a second step, and iii) defining a
narrow passageway that fluidly connects the inlet and outlet
regions in a fluid path, the narrow passageway including a first
passageway and a second passageway, the first passageway fluidly
connecting the inlet region and the second passageway, being
bounded by the first step and the surface of the void, and having a
height defined by the distance between the first step and the
surface of the void, and the second passageway fluidly connecting
the first passageway and the outlet region, being bounded by the
second step and the surface of the void, and having a height
defined by the distance between the second step and the surface of
the void, wherein the width of the narrow passageway at the portion
of the second step nearest the inlet region in the fluid path is
greater than the height of the second passageway, the height of the
second passageway being smaller than the height of the first
passageway, passing a first fluid from the inlet region into the
outlet region by way of the narrow passageway, whereby particles
are separated based on a characteristic dimension of the individual
particles, and thereafter collecting separated particles retained
within the void by way of passing a second fluid from the outlet
region into the inlet region by way of the narrow passageway.
2. The method of claim 1, wherein the first and second fluids have
substantially the same composition.
3. The method of claim 1, wherein the first fluid is passed from
the inlet region to the outlet region using a first fluid
displacement device to add the first fluid to the inlet region by
way of a fluid inlet port that extends through at least one of the
body and the cover and that fluidly communicates with the inlet
region during separation of the particles.
4. The method of claim 3, wherein the second fluid is passed from
the outlet region to the inlet region using a second fluid
displacement device to add the second fluid to the outlet region by
way of a fluid outlet port that extends through at least one of the
body and the cover and that fluidly communicates with the outlet
region during collection of separated particles.
5. The method of claim 4, wherein the first and second fluid
displacement devices are the same device.
6. The method of claim 3, wherein the second fluid is passed from
the outlet region to the inlet region by withdrawing fluid from the
inlet region by way of a fluid port that extends through at least
one of the body and the cover and that fluidly communicates with
the inlet region.
7. The method of claim 1, wherein the first fluid is passed from
the inlet region to the outlet region by withdrawing fluid from the
outlet region by way of a fluid port that extends through at least
one of the body and the cover and that fluidly communicates with
the outlet region.
8. The method of claim 7, wherein the second fluid is passed from
the outlet region to the inlet region using a second fluid
displacement device to add the second fluid to the outlet region by
way of a fluid outlet port that extends through at least one of the
body and the cover and that fluidly communicates with the outlet
region during collection of separated particles.
9. The method of claim 7, wherein the second fluid is passed from
the outlet region to the inlet region by withdrawing fluid from the
inlet region by way of a fluid port that extends through at least
one of the body and the cover and that fluidly communicates with
the inlet region.
10. The method of claim 1, wherein the particles are cells.
11. The method of claim 1, wherein the particles provided to the
inlet region are cells of a blood sample and the collected
particles are stem cells.
12. The method of claim 11, wherein the height of the second
passageway is sufficient to permit passage of blood platelets
therethrough.
13. The method of claim 11, wherein the blood sample is a cord
blood sample.
14. The method of claim 11, wherein the particles provided to the
inlet region are cells of a blood sample obtained from a pregnant
woman and the collected particles are fetal cells.
15. The method of claim 11, wherein the particles provided to the
inlet region are cells of a blood sample obtained from a pregnant
woman and the collected particles are fetal stem cells.
16. The method of claim 11, wherein the height of the first
passageway is about 12-16 micrometers and the height of the second
passageway is about 5.5 to 8.5 micrometers.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 13/170,369, filed 28 Jun. 2011, which is a
continuation of U.S. application Ser. No. 10/587,053 filed 11 Dec.
2006, which is now issued as U.S. Pat. No. 7,993,908 and was a 35
U.S.C. 371 filing of international application PCT/US02/22689,
filed 17 Jul. 2002, which is now inactive, and is also entitled to
priority pursuant to 35 U.S.C. .sctn.119(e) to U.S. provisional
patent application 60/306,296, which was filed on 17 Jul. 2001.
BACKGROUND OF THE INVENTION
[0002] Developments in methods of manufacturing very small devices,
such as microelectronic devices, have made it possible to precisely
and reproducibly make devices having features with nanometer-scale
dimensions. Apart from use of such methods in microelectronic
device production, similar technology has been used to make devices
for handling biological materials, such as cells and
macromolecules.
[0003] Microengineered bio-handling devices having structural
elements with minimal dimensions ranging from tens of micrometers
(the dimensions of biological cells) to nanometers (the dimensions
of some biological macromolecules) have been described. This range
of dimensions (nanometers to tens of micrometers) is referred to
herein as "microscale." For example, U.S. Pat. No. 5,928,880, U.S.
Pat. No. 5,866,345, U.S. Pat. No. 5,744,366, U.S. Pat. No.
5,486,135, and U.S. Pat. No. 5,427,946 describe microscale devices
for handling cells and biological molecules.
[0004] Hemocytometry is a field of medical analysis and research
wherein blood cells are analyzed using variety of techniques and
devices. Basic manually-operated devices such as microscope slides
with Neubauer or Makler chambers were developed over a century ago.
These devices are expensive, reusable, and lack flexibility,
multiple features, and disposability. Disposability is especially
desirable to minimize medical personnel interaction with
potentially hazardous biological specimens.
[0005] Every year, approximately 500,000 patients are diagnosed
with blood disorders worldwide, including about 30,000 per year in
the United States. Many blood disorders can be alleviated by
transplantation of stem cells (i.e., relatively non-differentiated
cells which retain at least hematopoietic capacity) into the
patient. The ideal source of stem cells is the same patient to whom
the cells are to be administered. However, hematopoietic (and
other) stem cells are relatively rare in adults, and can be
difficult to isolate in large numbers.
[0006] Blood drawn from the umbilicus shortly after delivery ("cord
blood") is a rich source of hematopoietic stern cells. Cord blood
storage methods are presently known and used commercially, but have
the drawback that a relatively large volume (e.g., 100 to 250
milliliters) of blood must be stored in order to preserve a
sufficient number of hematopoietic stem cells for use in future
medical procedures. The large volume of cord blood that is stored
increases the cost and decreases the convenience of the procedure.
The stored volume could be decreased significantly (e.g., to 0.1 to
1 milliliter) if stem cells could be separated from cord blood
prior to storage. However, present methods of separating stem cells
from cord blood are expensive and cumbersome and are sometimes
ineffective. The present invention overcomes the shortcomings of
previously known stem cell separation methods and facilitates
efficient and cost-effective separation of stem cells from cord
blood.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention relates to a microscale apparatus for
separating cells. The apparatus comprises a body, a cover, and a
separation element. The body defines avoid having inlet and outlet
regions. A separation region is interposed between the inlet and
outlet regions. The cover contacts the body and covers at least the
separation region of the void. The separation element is disposed
in the separation region of the void and contacts either the body
or the cover. The separation element has a plurality of flat
portions that are disposed at different distances from the cover or
body (respectively) to form steps or ramps. The highest step, the
walls of the void (i.e., the body) and the cover or body (depending
on the configuration) define a narrow passageway through which
fluid can pass from the inlet region to the outlet region. The
separation element can be attached to, or integral with, either the
body or the cover. The cover or the body can define one or more
(preferably at least two) fluid ports for providing liquid to, and
withdrawing liquid from, the void. Preferably, the cover, body, or
both, define a fluid inlet port in fluid communication with the
inlet region and a fluid outlet port in fluid communication with
the outlet region. A fluid displacement device can be attached to
one or both of the ports for providing fluid to the fluid inlet
port, withdrawing fluid from the fluid outlet port, or both.
Separate fluid handling devices can be attached to each of the
inlet and outlet ports.
[0008] The height of the narrow passageway, measured from the
separation element to the cover, depends on the characteristic
dimensions of the cells or particles that are to be retained or not
to be retained. For the apparatus described herein, the height of
the narrow passageway is generally in the range from 0.1 to 1000
micrometers (preferably in the range from 0.5 micrometer to 25
micrometers for animal or plant cells, or in the range 0.5 to 1.5
micrometers for bacteria).
[0009] The apparatus can have one or more fluid channels defined by
the body, the cover, the separation element, or some combination of
these. These fluid channels can be used to withdraw fluid from the
void at a step of the separation element. The apparatus can also
have a device for detecting a cell, a device for manipulating a
cell, or a device for killing a cell (e.g., a heating element) in
the void at a step of the separation element.
[0010] The apparatus can have a variety of surface modifications,
such as an antibody attached to one or more of a surface of the
separation element, a surface of the inlet region, a surface of the
outlet region, and a surface of the cover.
[0011] A plurality of the apparatus can be connected in series or
in parallel.
[0012] The invention also relates to a method of separating cells.
The method comprises providing the cells to the inlet region of an
apparatus of the type described herein and thereafter collecting
cells from one of a step of the separation element, the outlet
region, and the inlet region. Preferably, a fluid is passed from
the inlet region to the outlet region after (and/or while)
providing the cells to the inlet region, and the cells are
collected from a step of the separation element. For example, the
cells can be cells of a blood sample (e.g., a cord blood sample)
and the collected cells can be stem cells. When a blood sample is
used, the height of the narrow passageway is preferably sufficient
to permit passage of blood platelets therethrough.
[0013] The invention also includes a kit for separating cells. The
kit comprises the components of the apparatus described herein, and
can further comprise instructions or reagents for using the
apparatus.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0014] The foregoing summary, as well as the following detailed
description of preferred embodiments of the invention, will be
better understood when read in conjunction with the appended
drawings. For the purpose of illustrating the invention, there is
shown in the drawings embodiments which are presently preferred.
However, that the invention is not limited to the precise
arrangements and instrumentalities shown.
[0015] FIG. 1 is a cross section of a portion of the apparatus
described herein, showing the stepped structure of the separation
element. Numbers indicate relative distance between the separation
element (16) and the cover (12) or body (10).
[0016] FIG. 2 is a cross section of an example of an assembled
apparatus described herein, wherein the separation element (16) is
integral with the body (10), has steps on both the face facing the
inlet region and the face facing the outlet region, and has steps
(15) of various heights. A narrow passageway (18) exists between
the highest step and the cover (12). An inlet port (20) and an
outlet port (22) are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The invention relates to an apparatus for separating cells
on the basis of one or both of their size and geometry. The
apparatus comprises a body having a void therein. The void has an
inlet region and an outlet region and can be filled with fluid. A
separation element separates the inlet and outlet regions of the
void and has at least two steps with characteristic heights. A
cover is disposed across the void, and covers at least the portion
of the void wherein the highest portion of the separation element
occurs. Thus, fluid flow through the device passes from the inlet
region, across and over the separation element, and into the outlet
region.
[0018] In one embodiment, the cover is disposed across
substantially the entire area of the void, yielding a closed fluid
system. If fluid flow through the system is desired, the cover, the
body, or both, can have inlet and an outlet ports. The ports can be
simple holes which extend through the cover or body, or they can
have fixtures (burrs, rings, hubs, or other fittings) associated
with them for facilitating connection of a fluid handling device
with the port. These ports facilitate addition and withdrawal of
fluid and allow application of cells to the apparatus or collection
of cells therefrom. By way of example, the body can be adapted to
fit a peristaltic pump, a syringe pump, or substantially any other
fluid displacement device by way of Luer-type fittings.
[0019] The shape, material, and construction of the body are not
critical, except that the void in the body should be machined in
such a way that a cover can be applied across the void in order to
forma fluid-tight seal with the body around the edges of the void.
Preferably, the void is formed in a flat portion of the body, and a
flat cover is used that has a size and shape sufficient to
completely cover the void.
[0020] Similarly, the shape, material, and construction of the
cover are not critical, except that the cover should form a
fluid-tight seal with the body at the edges of the void and should
extend from the body at one edge of the void, along the highest
portion of the separation element across the void, to the body at
another edge of the void, thereby defining (with the separation
element) a narrow passageway through which liquid must flow in
order to pass from the inlet region to the outlet region of the
void.
[0021] The cover can be movable, bendable, or deflectable, in order
to change the size (i.e., height) and shape of the narrow
passageway if desired. Any of a variety of mechanical apparatus can
be used to accomplish movement of the cover (e.g., controllably
movable brackets having an inclined plane upon which the cover
rests, so that the cover can be lifted or lowered as desired).
Alternatively, the cover can have a bimetallic construction, so
that upon heating or applying an electrical potential to the two
metals, the cover bends. The direction of the bend can be predicted
using, for example, the thermal expansion coefficients of the two
metals, and a bimetallic cover can be arranged so that it decreases
or increases the size of the narrow passageway based on the
temperature at which it is maintained. As another alternative, the
cover can be manufactured from any of a variety of known
piezoelectric materials. Application of electrical potential to a
piezoelectric material induces deformation (i.e., bending) of the
material in ways that are predictable, depending on the identity of
the material.
[0022] The separation element has a stepped structure with at least
two steps. One of the steps is the highest. The separation element
can be attached to (or integral with) either the body or the cover,
or it can be a separate piece of material sandwiched between the
body and the cover. When the device is assembled, the steps of the
separation element define selected distances between the separation
element and either the body (i.e., the surface of the void in the
body) or the cover. Assessed in the direction from the inlet region
of the void toward the outlet region of the void, the distance
between the steps and the body or cover decreases. Thus, movement
of cells suspended in a fluid flowing through the assembled
apparatus can be inhibited or halted at a step that is
characteristic of a dimension of the cell (e.g., the diameter of a
substantially spherical cell or the rotational diameter of an
irregularly-shaped cell).
[0023] In the same manner, substantially any particles that have
characteristic dimensions on the same order as the distance between
the steps and the cover or body can be separated using the
apparatus. For example, bacteria can be sorted and separated using
an apparatus that has a separation element that has steps disposed
from the cover or body in increments of tenths of micrometers
(e.g., 0.5 to 1.5 micrometers). Similarly, mineral particles and
other small inanimate objects can be sorted using apparatus
comprising a separation elements having steps which vary on the
order of a characteristic dimension of the objects. Known
fabrication techniques enable fabrication of steps which Wily by
tenths of micrometers to thousands of micrometers. At a micrometer
scale, as preferred for the apparatus described herein, the height
of the narrow passage is generally not more than 1 millimeter, and
the steps vary in height by tenths of micrometers, micrometers,
tens of micrometers, hundreds of micrometers, or combinations of
these (e.g., see FIG. 2).
[0024] Cells which have a characteristic dimension that is smaller
than the height of the narrow passageway will pass through the
device from the inlet region to the outlet region. Likewise, cells
which are capable of deforming to fit through the narrow passageway
can pass from the inlet region to the outlet region. If the
separation element has multiple steps, then movement of cells
through the apparatus will be halted at a step that is separated
from the cover or body by a distance that is smaller than a
characteristic dimension of the cell. Using such a separation
element, cells of several different types can be concentrated at
different portions of the apparatus.
[0025] The steps of the separation element can be discrete steps,
as illustrated in FIG. 1. Alternatively, one or more of the steps
can be sloped, the steps being separated from one another by a flat
portion that extends in the direction from the inlet region toward
the outlet region. The length (in the direction of fluid flow) of
the flat portion is not critical. However, where cells having a
particular characteristic dimension are anticipated to be applied
to the device, it can be preferable to include a relatively long
flat portion before the first step that is expected to inhibit or
prevent flow of the cells with the fluid, so that the cells can
accumulate on that step. The flat portions of different steps can
have the same length, or they can have different lengths, as
illustrated in FIG. 2.
[0026] The heights of the steps can be selected based on the
expected characteristic dimensions of cells in samples of a type
intended to be applied to the apparatus. Alternatively, the heights
of the steps can be selected arbitrarily. Thus, steps can differ in
height by as little as 1 or a few nanometers or by as much as a
few, several, or even tens of micrometers. The steps must occur on
the face of the separation element that faces the inlet region of
the void, so that cells moving from the inlet region toward the
outlet region must traverse the steps. The steps can also occur on
the face of the separation element that faces the outlet region of
the void if this facilitates manufacture of the apparatus, or so
that the apparatus can be used in either an inlet-to-outlet or
outlet-to-inlet flow configuration. When the separation element has
steps on the face that faces the outlet region, then cells which
pass through the narrow passageway can be sorted on the trailing
edge of the separation element. For example, many cells exhibit at
least limited deformability, and some cells will often pass through
the narrow passageway even though their characteristic dimensions
indicate that they should not (i.e., some cells which appear too
large to pass through the narrow passageway nonetheless pass). If
these cells settle on a step on the outlet side of the separation
element, then they can be collected, detected, observed, ablated,
or manipulated, as with cells on a step on the inlet face of the
separation element.
[0027] The body, the cover, or both, can have one or more fluid
channels that fluidly connect with the surface of a step of the
separation element, for removing fluid from the step (including any
cells suspended in the fluid upon that step). Furthermore, when the
step has discrete grooves or regions in the step, the cover or body
can be machined so that the fluid channels fluidly communicate most
nearly with a discrete groove or region upon the step, for removing
fluid in the vicinity of that groove or region of the step.
Likewise, the body, the cover, the separation element, or some
combination of these, can have an optical, electrical, or
optico-electrical device constructed therein or thereon (e.g., by
etching, film deposition, or other known techniques) at a position
that corresponds to a selected step or a selected groove or region
of a step. Such devices can be used to detect cells (e.g., using a
detector to detect a decrease in light or other radiation
transmitted across the fluid between the surface of the step and
the cover or body) or to manipulate cells (e.g., using an
activatable heating element to ablate cells which pass or rest near
the heating element). Devices constructed upon the cover, the body,
or the steps can be made individually activatable by assigning an
electronic address to the device. In this manner, cells can be
detected at discrete areas of the device, and cells at selected
areas can be manipulated without manipulating cells at other
positions.
[0028] Harvesting of cells from a selected step (or a plurality of
selected steps) can be performed by simply withdrawing fluid from
that step or a portion of the step. In some instances, such as when
adhesion between cells and a step upon which they rest occurs, it
can be advantageous to apply energy to the apparatus in order to
dislodge the cells or otherwise facilitate their removal. The
energy can be applied in many forms, and a preferable form will
usually depend on the type of cell or object to be displaced and
the identity of the force or phenomenon which inhibits removal of
the cell or object from the step. By way of example, withdrawal of
fluid from one portion of a step can be performed simultaneously
with addition of fluid at another portion of the same step. Other
examples of forms in which energy can be applied to the apparatus
in order to harvest cells include shaking, tapping, or vibrating
the apparatus, or applying energy in the form of ultrasound, heat,
infrared or other radiation, bubbles, compressed air, and the
like.
[0029] Instead of recovering cells that are retained on one or more
steps of the separation element, the cells can instead be detected
or manipulated. In one embodiment, one or more cells are lysed by
application to the cells of electrical, mechanical, or heat energy,
thereby releasing the contents of the cell in the void of the
apparatus. The cell contents can be analyzed or manipulated in the
apparatus, or they can be recovered from the apparatus and analyzed
or manipulated outside of the apparatus. By way of example, a cell
that is retained at a particular location on a step can be lysed
using a device located at that particular location, thereby
releasing the cell's DNA into the void. The DNA can be amplified in
the void by providing PCR reagents to the void, or it can be
collected (e.g., by passing fluid through the void and collecting
the DNA in the outlet fluid) and amplified outside of the
apparatus. The apparatus can thus be used to analyze the contents
of individual cells or groups of cells.
[0030] When the apparatus is filled with fluid, the fluid fills the
void, including the inlet and outlet regions, and completely covers
the separation element. If desired, the apparatus can be lightly
manipulated (e.g., by tapping or shaking), or the fluid can be
applied under pressure, in order to ensure that all portions of the
void are filled with the fluid and to remove any air bubbles that
may be present. Air bubbles, if present, can also be removed by
passing fluid from the inlet region to the outlet region.
[0031] The body, cover, and separation element can be constructed
from substantially any material that will hold its shape during
operation of the apparatus as described herein. However, rigid
materials are preferred. Examples of suitable materials include
various glasses, solid polymers, and crystalline minerals. Silicon
is a preferred substrate material because of the well-developed
technology permitting its precise and efficient fabrication, but
other materials can be used, including various glasses and cast,
molded, or machined polymers including polytetralluoroethylenes.
The inlet and outlet ports, the separation element, and the
surfaces defining the void in the body can be fabricated
inexpensively in large quantities from a silicon substrate by any
of a variety of micromachining methods known to those skilled in
the art. The micromachining methods available include film
deposition processes such as spin coating and chemical vapor
deposition, laser fabrication or photolithographic techniques such
as UV or X-ray processes, precision machining methods, or etching
methods which may be performed by either wet chemical processes or
plasma processes. (See, e.g., Manz et al., 1991, Trends in
Analytical Chemistry, 10:144-149).
[0032] Steps of varying widths and heights can be fabricated with
microscale dimensions for separating cells in a sample. A silicon
substrate containing a fabricated steps can be covered and sealed
(e.g., anodically bonded) with a thin glass cover. Other clear or
opaque cover materials may be used. Alternatively, two silicon
substrates can be sandwiched, or a silicon substrate can be
sandwiched between two glass covers. Preferably, at least one of
the body and the cover is transparent. Use of a transparent
material facilitates dynamic viewing of the contents of the device,
and allows probing of fluid flow in the apparatus, either visually
or by machine. Other fabrication approaches can be used.
[0033] One advantage of the apparatus described herein is that they
can be manufactured in a wide variety of sizes and geometrical
arrangements, depending on the intended use of the apparatus. In
addition, multiple apparatus can be manufactured on or in a single
piece of material, such as a unitary silicon block or a microscope
slide. Furthermore, the multiple apparatus on a slide can be
connected in series, in parallel, or both. By way of example,
several apparatus having a narrow passageway of relatively small
height (e.g., 2 micrometers) can be constructed on a single block
of material, and a sample (e.g., cord blood) can be fed to the
inlet region of each of those apparatus. By feeding fluid through
the apparatus, platelets and small cell debris can be removed from
the sample. Stem cells and red and white blood cells will be
retained in the inlet region or on the steps.
[0034] After platelets have been removed, the flow in the apparatus
can be reversed, and the retained cells can be transferred
(directly, by way of fluid circuit switching, or indirectly, by
manually or automatically collecting the cells and reapplying them)
to the inlet region of one or more second apparatus. These second
apparatus can, but need not, be made in the same piece of material
as the first series of apparatus. These second apparatus comprise a
separation element that separates stem cells from red blood cells
based on their different characteristic dimensions.
[0035] By feeding fluid through the second apparatus, red blood
cells are separated from stem cells. If desired, the narrow
passageway of the second apparatus can have dimensions that permit
passage therethrough of the red blood cells. Stem cells can be
collected from the second apparatus, for example by way of a fluid
channel machined into the body at a position where accumulation of
stem cells is anticipated. If desired, the collected stem cells can
be provided to the inlet region of a third apparatus which
comprises a separation element having a cell-type specific antibody
attached to one or more steps thereof (or a portion of one or more
steps), in order to separate stem cells from remaining non-stem
cells, or to separate different classes of stem cells.
[0036] The capacity of the apparatus described herein depends on
the number of cells that can occupy a step of the separation
element without occluding or severely limiting fluid flow. Because
passage of a cell past a step of the separation element depends
primarily upon the distance between the step and the cover or body,
the width of the step (and thus the width of the narrow passageway)
is not critical. Advantageously, the steps can be made very wide or
many steps can be arranged in parallel. Thus, if one portion of a
step becomes clogged with cells that cannot fit in the space
between the step and the cover or body, fluid flow can continue
along the remaining width of the step. In one embodiment, the steps
are arranged concentrically in a circular, oval, rippled or
irregular form, so that the width of steps upon which cell
accumulation is anticipated can be maximized.
[0037] The surfaces of the apparatus can be chemically treated or
coated with any of a variety of known materials which reduce or
enhance agglutination of cells with the material selected for the
cover, body, or obstacles. By way of example, an antibody which
binds specifically with a cell-surface antigen can be attached to a
surface of the void using any of a variety of protein anchoring
chemistries, a surface of a step, or a surface of the cover, in
order to inhibit passage of cells which exhibit the antigen past
the surface (e.g., in order to differentiate cells of similar size
but different type). The surfaces of the apparatus can also be
treated with any of a variety of known reagents (e.g., oxygen
plasma) in order to increase the hydrophilicity of the surfaces.
This treatment can improve the rate and completeness of filling of
the apparatus with a fluid medium introduced into the
apparatus.
[0038] The assembled apparatus can be used to separate cells by
providing cells to the inlet region of the void. If the cells are
motile, then they can distribute themselves on the steps of the
separation element. More typically, however, the apparatus will be
used by applying a fluid flow between the inlet and outlet regions
of the void (e.g., by way of fluid ports fluidly connected with
each). Cells can be provided to the inlet region in fluid flowing
through the apparatus, by a separate port, or by providing the
cells to the region prior to applying the cover to the body. Fluid
flow carries or pushes the cells in the direction from the inlet
region toward the separation element. If a cell has a
characteristic dimension smaller than or equal to the distance
between the first step and the cover or body, then the cell can be
carried onto the first step. Similarly, if the characteristic
dimension of the cell is smaller than or equal to the distance
between the next step and the cover or body, then the cell can be
carried onto the next step. If the cell encounters a step for which
the distance between the step and the step and the cover or body is
smaller than the characteristic dimension of the cell, then the
cell cannot pass onto the step unless the cell can deform to fit
through the gap. Celts that are retained on a step can be collected
or manipulated using fluid channels or devices that are addressed
to the particular step.
[0039] In one embodiment, the apparatus is made to separate stem
cells from other cells in a blood sample (e.g., a cord blood
sample). Stem cells are generally larger (70% have a characteristic
dimension of about 12-16 micrometers) than red blood cells (which
are flat bi-concave disc-shaped cells having characteristic
dimensions of about 5.5 to 8.5 micrometers in diameter and 1.2-1.9
micrometers thick) or platelets (characteristic dimension=about
micrometer). This same apparatus can be used to separate fetal
cells from its mother's peripheral blood cells in order to analyze
the fetal cells (e.g., for indications of likely birth defects).
Fetal cells are known to circulate in the mother's bloodstream.
[0040] If a device is used in which the height of the narrow
passageway is more than about 1 micrometer, then most platelets
wilt pass through the device without being retained on the
separation element. If the height of the narrow passageway is about
2-5 micrometers, then most red blood cells, white blood cells, and
stern cells will be retained on one or more steps of the separation
element. If the separation element includes steps that are
sufficiently distant/e.g., 2 to 10 micrometers) from the cover or
body to permit red blood cells to pass upon the step, but which are
not sufficiently distant to permit stem cells to pass upon the
step, then the stem cells will be separated from the red blood
cells.
[0041] If the width, length, or both, of steps which accommodate
red blood cells are sufficiently great, then substantially all red
blood cells in the applied sample can be collected on those steps,
and substantially all stem cells will be excluded from those steps
and will remain in the inlet region or at steps having a greater
distance between the step and cover or body (e.g., more than 12
micrometers). Collection of fluid from the inlet region or from one
or more steps which accommodate stem cells can result in isolation
of the stem cells from red blood cells of the blood sample. The
collected stem cells can be centrifuged, ultrafiltered, or
otherwise compacted into a form convenient for storage.
[0042] The net effect of this procedure can be to dramatically
reduce the volume of the blood sample which must be stored in order
to store the stem cells contained therein. This procedure also
isolates the stern cells from red blood cells and from
iron-containing compounds (e.g., proteins such as ferritin and
hemoglobin) that could potentially harm the stem cells or induce an
immune response in a recipient of the blood sample.
[0043] The cover, body, and separation element (if not already
connected to one of the cover and body) can be provided in the form
of a kit to be assembled by the user (e.g., after adding a fluid
medium to the void in the body). The kit can also include
instructions for using the apparatus or reagents to be used
therewith. The apparatus can be supplied pre-filled with fluid.
[0044] The apparatus can have indicia associated in a fixed
position with respect to the separation element. The indicia can be
used to assess whether cells having a selected characteristic
(e.g., size or ability to bind with an antibody fixed to a surface
of the apparatus) are being retained in the apparatus. The indicia
can be printed, painted, or stamped on, or engraved or etched in
the body or the cover, preferably on a surface of a component that
is transparent, so that the indicia and the cells in the apparatus
can be simultaneously observed by a user. The indicia preferably do
not alter the shape, diameter, or smoothness of the fluid path with
which they are associated. For example, the indicia can be on or in
the opposite face of a transparent material in which the fluid path
exists. Alternatively, the indicia can be on or in one face of a
transparent material that has a different face opposed against the
fluid path (e.g., the exterior face of the cover).
[0045] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but covers modifications within
the spirit and scope of the present invention as defined by the
appended claims.
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