U.S. patent application number 12/155643 was filed with the patent office on 2008-10-02 for method and device for manipulating individual small objects.
This patent application is currently assigned to Seng Enterprises Ltd.. Invention is credited to Mordechai Deutsch.
Application Number | 20080241874 12/155643 |
Document ID | / |
Family ID | 30012047 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241874 |
Kind Code |
A1 |
Deutsch; Mordechai |
October 2, 2008 |
Method and device for manipulating individual small objects
Abstract
A method for individually moving small objects, such as cells,
from one location to another as well as a device for implementing
the method is disclosed. A small object such as a cell is isolated
at some initial location and moved to some destination location by
the movement of the small object through a succession of
intermediate locations until the small object arrives at the
destination location. Also disclosed are methods of manipulating
cells made possible by the method and device of the present
invention.
Inventors: |
Deutsch; Mordechai; (Moshav
Olesh, IL) |
Correspondence
Address: |
Martin D. Moynihan;PRTSI, Inc.
P.O. Box 16446
Arlington
VA
22215
US
|
Assignee: |
Seng Enterprises Ltd.
Larnaca
CY
|
Family ID: |
30012047 |
Appl. No.: |
12/155643 |
Filed: |
June 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10546784 |
Aug 25, 2005 |
7405071 |
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PCT/IL2004/000194 |
Feb 26, 2004 |
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12155643 |
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60496382 |
Aug 20, 2003 |
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60496383 |
Aug 20, 2003 |
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60523094 |
Nov 19, 2003 |
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60523096 |
Nov 19, 2003 |
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Current U.S.
Class: |
435/29 ;
435/173.9 |
Current CPC
Class: |
B01L 2300/0819 20130101;
B01L 3/502761 20130101; B01L 2400/0415 20130101; B01L 2300/0838
20130101; B01L 2400/043 20130101; B01L 2200/0668 20130101; G01N
15/1404 20130101; B01L 2400/0487 20130101; G01N 2015/1415 20130101;
G01N 2015/149 20130101; B01L 2400/0463 20130101 |
Class at
Publication: |
435/29 ;
435/173.9 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12N 13/00 20060101 C12N013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2003 |
IL |
154677 |
Claims
1-67. (canceled)
68. A method of moving an individual small object from an initial
location to a destination location, comprising: a. defining a
series of N+1 locations L.sub.0 . . . L.sub.N, N being an integer
greater than 1 wherein L.sub.0 is the initial location and L.sub.N
is the destination location; and b. for i from 0 to N-1,
successively moving the individual small object from a location
L.sub.i to a location L.sub.i+1 wherein said moving is effected by
applying an attractive force from said location L.sub.i+1 to the
individual small object so as to cause the individual small object
to move from a said location L.sub.i to a said location L.sub.i+1,
and wherein the order of the size of the individual small object is
equal to or less than 10.sup.3 micron.
69. The method of claim 68, wherein said location Li is fixed
relative to said location Li+1.
70. The method of claim 68, further comprising applying a repulsive
force to the individual small object from location L.sub.i.
71. The method of claim 70, wherein said repulsive force is
selected from the group consisting of physical, electrical and
magnetic forces.
72. The method of claim 70, wherein said repulsive force is a
result of a flow of fluid from said location L.sub.i.
73. The method of claim 72, wherein said flow of fluid is generated
by injection of a fluid from the vicinity of location L.sub.i.
74. The method of claim 68, wherein said attractive force is
selected from the group consisting of physical, electrical and
magnetic forces.
75. The method of claim 68, wherein said attractive force is a
result of a flow of fluid towards said location L.sub.i+1.
76. The method of claim 75, wherein said flow of fluid is generated
by suction of a fluid from the vicinity of location L.sub.i+1.
77. The method of claim 68, wherein at least one of said locations
L.sub.0 . . . L.sub.N, comprises an enclosure of a size sufficient
to substantially enclose the individual small object.
78. The method of claim 68, wherein said fluid is a liquid.
79. The method of claim 68, wherein said liquid is a cell culture
medium.
80. A method of moving an individual small object in a fluid from a
first location to a second location, comprising: a. drawing fluid
towards the first location so as to produce a first force
localizing the small object at the first location; and b.
subsequently drawing fluid towards the second location so as to
produce a second force localizing the small object at the second
location, wherein the order of the size of the individual small
object is equal to or less than about 10.sup.3 micron.
81. The method of claim 80, wherein said first location is fixed
relative to said second location.
82. The method of claim 80, wherein said drawing of fluid towards
the first location and towards the second location is performed by
suction of the fluid by a flow generator.
83. The method of claim 80, further comprising reducing the
magnitude of said first force so as to assist in said localizing of
the small object at the second location.
84. The method of claim 80, further comprising injecting fluid from
the first location so as to produce a repulsive force to assist in
said localizing of the small object at the second location.
85. The method of claim 80, wherein said fluid is a liquid.
86. The method of claim 85, wherein said liquid is a cell culture
medium.
87. A method of studying a cell, comprising suspending said cell
individually in a liquid by using a Bernoulli effect.
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of
micromanipulation and, more particularly, to a method to and device
for physically manipulating small objects, especially living cells.
The present invention also relates to the field of life sciences,
and more particularly to a device and methods for individually
manipulating cells.
[0002] In the fields of biology and medicine it is often important
to study the results of the interaction between living cells and
external factors such as environment, physical stimuli, chemical
stimuli and other cells. Such studies are important, for example,
for medical diagnostics, drug development and the understanding of
basic cell function and are generally divided into two types,
static and dynamic studies.
[0003] In static studies a group of cells are exposed to whatever
stimulus is being studied and, after a certain time, the magnitude
and nature of the reaction of the group of cells to the stimulus
are determined.
[0004] In dynamic studies a single cell or a group of cells is
exposed to whatever stimulus is being studied and the magnitude and
nature of the reaction of the cell or group to the stimulus are
determined, either continuously or discretely.
[0005] Both static and dynamic studies are often performed on a
population of cells on a macroscopic scale, in cuvettes or well
arrays. One disadvantage of studying populations of cells is that
the results may teach of secondary effects rather than primary
effects of stimuli: the response of a cell to the response of other
cells to a stimulus is detected rather than the effect of the
stimulus itself. Another disadvantage of such studies is that the
results reflect a distribution of effects on a group of cells. This
disadvantage is partially overcome by the use of homogenous
populations of cells. Although the study of homogenous populations
of cells is informative, results are not necessarily representative
of "real-life" effects. Although theoretically possible, the
required manipulations using available technologies are too
difficult and too slow when applied to the study of individual
cells.
[0006] Static and dynamic studies are also performed using flow
cytometry. In flow cytometry, the response of a cell to a stimulus
is measured or detected for a single cells moving through a medium,
the cell being discarded subsequently to the measurement.
Flow-cytometry allows neither the study of an individual cell over
a period of time nor the study of the interaction of two or more
specifically selected cells.
[0007] Practitioners of flow cytometry realize that many critical
questions in cell biology, developmental biology, immunology,
oncology, pharmacology and virology cannot be answered using even
the most sophisticated flow cytometry techniques. It is clear to
one skilled in the art that complete understanding of complex
biological problem requires the dynamic manipulation and study of
single cells or of a multiplicity of cells studied as individuals
in parallel or sequentially.
[0008] There is thus a widely recognized need for, and it would be
highly advantageous to have, a method to manipulate, isolate and
study single, whole, living cells as individuals.
SUMMARY OF THE INVENTION
[0009] The above needs are met by the present invention which
allows isolation and manipulation of an individual small object
such as a single cell.
[0010] The present invention successfully addresses the
shortcomings of the prior art by providing a method for moving a
small object (such as a cell) from one location to another
location, thus allowing isolation and manipulation of the small
object. The present invention also provides a device for
implementing the method of the present invention.
[0011] According to the teachings of the present invention there is
provided a method of moving an individual small object from an
initial location to a destination location by: [0012] a. defining a
series of N+1 locations L.sub.0 . . . L.sub.N, N being an integer
greater than 1 wherein L.sub.0 is the initial location and L.sub.N
is the destination location; and [0013] b. for i from 0 to N-1,
successively moving the individual small object from a location
L.sub.i to a location L.sub.i+1 wherein moving the small object is
effected by applying an attractive force from location L.sub.i+1 to
the individual small object so as to cause the individual small
object to move from location L.sub.i to location L.sub.i+1.
[0014] According to an embodiment of the present invention, the
position and/or orientation of location L.sub.i is fixed relative
to the position and/or orientation of location L.sub.i+1.
[0015] According to an embodiment of the present invention, to aid
in moving the small object, a repulsive force is applied to the
individual small object from location L.sub.i before, during or
after application of the attractive force from location
L.sub.i+1.
[0016] According to a feature of the present invention, either or
both an attractive force or a repulsive force can be physical,
electrical and/or magnetic.
[0017] According to a preferred embodiment of the present
invention, a repulsive force is a result of a flow of fluid from
location L.sub.i, preferably the flow of fluid being generated by
injection of a fluid from the vicinity of location L.sub.i.
[0018] According to a preferred embodiment of the present
invention, an attractive force is a result of a flow of fluid
towards location L.sub.i+1, preferably the flow of fluid being
generated by suction of a fluid from the vicinity of location
L.sub.i+1.
[0019] According to a feature of the present invention, at least
one (and preferably all) of the locations L.sub.0 . . . L.sub.N,
includes an enclosure of a size sufficient to substantially enclose
(partially or completely) the individual small object (and
preferably no more than one small object at a time).
[0020] According to the teachings of the present invention there is
also provided a method of moving an individual small object in a
fluid from a first location to a second location by: [0021] a.
drawing fluid towards the first location so as to produce a first
force localizing the small object at the first location; and [0022]
b. subsequently drawing fluid towards the second location so as to
produce a second force localizing the small object at the second
location.
[0023] According to an embodiment of the present invention, the
position and/or orientation of the first location is fixed relative
to the position and/or orientation of the second location.
[0024] According to an embodiment of the present invention, the
drawing of fluid towards the first location and the drawing of
fluid towards the second location are performed by suction of the
fluid by a flow generator. Suitable flow generators include, but
are not limited to, one or more pumps, and systems that use valves
and the like.
[0025] According to an embodiment of the present invention, before,
during or after the drawing of fluid towards the second location,
the magnitude of the first force is reduced so as to assist in
localizing the small object at the second location.
[0026] According to an embodiment of the present invention, before,
during or after the drawing of fluid towards the second location,
the first force is eliminated so as to assist in localizing the
small object at the second location.
[0027] According to an embodiment of the present invention, before,
during or after the drawing of fluid towards the second location,
fluid is injected from the first location so as to produce a
repulsive force to assist in localizing the small object at the
second location.
[0028] According to an embodiment of the present invention, the
first location and the second location include an enclosure of a
size sufficient to substantially enclose (partially or completely)
the individual small object (and preferably no more than one small
object at a time). In such an embodiment, the drawing of fluids
towards the first location and towards the second location is
preferably performed by suction of the fluids from within a
respective enclosure.
[0029] Depending on the specific embodiment of the present
invention, the size of individual small objects manipulated
according to the teachings of the present invention include objects
is in the order of equal to or less than about 10.sup.3 micron,
equal to or less than about 10.sup.2 micron, equal to less than
about 10.sup.1 micron or equal to less than about 10.sup.0 micron.
Such objects include crystals, bacteria, viruses, proteins,
polymers, macromolecules, ions and atoms, but especially cells.
[0030] In embodiments of the present invention using a fluid, it is
preferred that the fluid is a liquid, preferably a cell culture
medium.
[0031] According to the teachings of the present invention there is
also provided a cell manipulation device, specifically configured
to manipulate individual cells found in a liquid such as a cell
culture medium. The cell manipulation device according to the
teachings of the present invention includes: [0032] a. at least N
locations; and [0033] b. at least two flow generators, each flow
generator being functionally associated with a location; wherein N
is at least two; wherein at least two of the flow generators are
independently settable to a mode selected from the group of modes
including a suction mode and an inactive mode; and wherein at least
two of the independently settable flow generators are each
associated with a different location. One embodiment of a device of
the present invention has two locations, each one of the two
locations having a functionally associated independently settable
flow generator.
[0034] According to an embodiment of the present invention, the
position and/or orientation of at least one of the N locations is
fixed relative to the position and/or orientation of another one of
the N locations. According to a preferred embodiment of the present
invention, all of the at least N locations having a fixed relative
position and/or orientation relative to the other locations.
[0035] According to an embodiment of the present invention, N is at
least three and at least three of the at least N locations are
arranged in a one-dimensional array. By one-dimensional array is
meant that each one of the N locations is adjacent to no more than
two other locations.
[0036] According to an embodiment of the present invention, N is at
least four and at least four of the at least four locations are
arranged in a two-dimensional array. By two-dimensional array is
meant that at least one of the N locations is adjacent to more than
two other locations.
[0037] According to an embodiment of the present invention, N is at
least five and at least five of the at least five locations are
arranged in a rectangular lattice array. By rectangular lattice
array is meant that at least one of the N location is adjacent to
four other locations. Preferably, substantially all "non-border"
locations are adjacent to four other locations.
[0038] According to an embodiment of the present invention, N is at
least seven and at least seven of the at least seven locations are
arranged in a hexagonal lattice array. By hexagonal lattice array
is meant that at least one of the N location is adjacent to six
other locations. Preferably, substantially all "non-border"
locations are adjacent to six other locations.
[0039] According to various embodiments of the present invention, N
is at least ten, 19, 24, 36 and even more.
[0040] According to an embodiment of the present invention, at
least one of the flow generators is independently settable to a
mode selected from the group of modes including a suction mode, an
injection mode and an inactive mode. Preferably, substantially all
of the flow generators are independently settable to a mode
selected from the group of modes including a suction mode, an
injection mode and an inactive mode.
[0041] According to an embodiment of the present invention, at
least one of the N locations is associated with at least two flow
generators and each one of these at least two flow generators is
independently settable to a mode selected from the group of modes
including a suction mode and an inactive mode (and preferably a
suction mode, an injection mode and an inactive mode).
[0042] According to an embodiment of the present invention, at
least one of the N locations is associated with at least three flow
generators and each one of these at least three flow generators is
independently settable to a mode selected from the group of modes
including a suction mode and an inactive mode (and preferably a
suction mode, an injection mode and an inactive mode).
[0043] According to an embodiment of the present invention, at
least two of the at least N locations are substantially defined by
the presence of an inlet of a respective flow-generator.
[0044] According to an embodiment of the present invention, at
least two of the at least N locations are substantially depressions
in a surface.
[0045] According to an embodiment of the present invention, at
least two of the at least N locations are substantially open
trapping-ends of enclosures. Each such enclosure is of a size
sufficient to substantially enclose (partially or completely) a
cell (and preferably no more than one cell). According to a
preferred embodiment, within at least two of the at least two
enclosures emerges an inlet of a respective flow generator. The
device of the present invention is specifically configured to
manipulate cells. Accordingly, the size of open trapping ends of
enclosures is of a size sufficient to allow entry of a cell to be
studied, and preferably only one such cell at a time. Accordingly,
according to embodiments of the present invention, the order of the
size of the open trapping ends are equal to or less than about
10.sup.3 micron, equal to or less than about 10.sup.2 micron, equal
to or less than about 10.sup.1 micron and/or equal to or less than
about 10.sup.0 micron.
[0046] According to an embodiment of the present invention, to
allow for efficient transfer of cells between one location and
another location (in accordance with the method of the present
invention), at least one location is no more than 1000 micron
distant from at least one other location, preferably substantially
every location being no more than 1000 micron distant from at least
one other location.
[0047] According to an embodiment of the present invention, to
allow for efficient transfer of cells between one location and
another location (in accordance with the method of the present
invention), at least one location is no more than 100 micron
distant from at least one other location, preferably substantially
every location being no more than 100 micron distant from at least
one other location.
[0048] According to an embodiment of the present invention, to
allow for efficient transfer of cells between one location and
another location (in accordance with the method of the present
invention), at least one location is no more than 10 micron distant
from at least one other location, preferably substantially every
location being no more than 10 micron distant from at least one
other location.
[0049] According to an embodiment of the present invention, to
allow for efficient transfer of cells between one location and
another location (in accordance with the method of the present
invention), at least one location is no more than 1 micron distant
from at least one other location, preferably substantially every
location being no more than 1 micron distant from at least one
other location.
[0050] According to an embodiment of the present invention, at
least one of the locations is configured so as to suspend a cell at
distance above itself using a Venturi effect, as explained
hereinbelow in detail.
[0051] According to an embodiment of the present invention, at
least one of the locations is provided with a cell wall penetrating
tool. According to a feature of the present invention, each such
cell wall penetrating tool is a member (including but not limited
to needles, wires and sharpened protuberances) pointing in a
direction substantially opposite a flow generated by a flow
generator functionally associated with the location when set to a
suction mode. As will be described hereinbelow in detail, when the
flow generator is set to a suction mode, a cell is pulled towards
and onto the cell wall penetrating tool with sufficient force to
effect cell wall penetration.
[0052] According to the teachings of the present invention there is
also provided a method of studying a cell by suspending the cell
individually in a liquid by using a Venturi effect.
[0053] According to the teachings of the present invention there is
provided a device for suspending a cell in a liquid including: a. a
substantially hollow body having an open trapping-end and a central
axis; b. emerging within the hollow body, at least one outlet of a
flow generator configured to inject liquid into the hollow body and
through the open trapping-end in a first flow substantially
parallel to the central axis of the hollow body; and c. emerging
within the hollow body, at least one inlet of a flow generator
configured to draw liquid through the open trapping-end and out of
the hollow body in a second flow substantially parallel to the
central axis of the hollow body; wherein the first flow is closer
to the central axis than the second flow. Such a cell suspension
device can be integrated into the cell manipulation device of the
present invention, where the hollow body defines, in part, a
location or enclosure as described hereinabove.
[0054] According to the teachings of the present invention there is
also provided a method of penetrating a cell wall by: a. immersing,
in a liquid, a cell wall penetrating tool pointing in a first
direction; and b. applying suction from a second direction opposite
the first direction so as to cause a cell in the liquid to be
carried against the cell wall penetrating tool where the intensity
of the carrying is sufficient to cause a wall of the cell to be
penetrated.
[0055] According to the teachings of the present invention there is
also provided a device for penetrating a cell wall including a. a
substantially hollow body having an open trapping-end; b. emerging
within the hollow body, at least one inlet of a flow generator
configured to draw liquid out of the hollow body in a flow having a
first direction; and c. a cell wall penetrating tool pointing in a
second direction substantially opposite the first direction. Such a
cell wall penetrating device can be integrated into the cell
manipulation device of the present invention, where the hollow body
defines, in part, a location or enclosure as described
hereinabove.
[0056] According to the teachings of the present invention there is
also provided a method for selecting cells by: a. providing a group
of cells; b. isolating cells from the group of cells in an
individual enclosure; c. examining each isolated cell; and d.
selecting isolated cells fulfilling at least one criterion.
According to a feature of the present invention, the selected cells
are separated from the group of cells.
[0057] According to the teachings of the present invention there is
also provided a method for treating a cell-containing biological
fluid by a. providing the cell-containing biological fluid; b.
identifying cells to be treated in the biological fluid; c.
differentiating between cells to be treated and other cells; and d.
either or both:
[0058] i. physically separating the cells to be treated from the
other cells; and/or
[0059] ii. directly manipulating the cells to be treated thus
treating the biological fluid.
[0060] According to a feature of the present invention, during the
identifying, each cell is isolated in an individual enclosure.
[0061] Examples of direct manipulation of the cells to be treated
includes but is not limited to destroying the cells to be treated
(e.g., physically, chemically, irradiation), exposing the cells to
be treated to a reagent (e.g. internally or externally, chemical
reagents, biological reagents), exposing the cells to be treated to
radiative energy (e.g. ultraviolet, infrared, visible light,
coherent light, incoherent light, microwaves) and penetrating the
cell walls of the cells to be treated (e.g. to inoculate the cells
or to remove a sample from therewithin).
[0062] Examples of treatable biological liquids include but are not
limited to lymphatic fluids, blood, cerebral spinal fluids, semen,
saliva, synovial fluid, bone marrow, cochlear fluid, fluid
extracted from tumors, ovarian fluid, amniotic fluid and chorionic
fluid.
[0063] According to an embodiment of the present invention,
subsequent to d, the biological liquid is directed into a living
organism (both human and non-human).
[0064] According to an embodiment of the present invention,
providing the biological liquid includes a step of taking the
biological liquid from a living organism (both human and
non-human).
[0065] According to an embodiment of the present invention, e.
subsequently to d, the biological liquid is directed into a living
organism (both human and non-human) and providing the biological
liquid includes a step of directing the biological liquid to flow
from the living organism to the individual enclosures.
[0066] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention, suitable methods and materials
are described below. All publications, patent applications, patents
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, embodiments and examples are illustrative only and not
intended to be limiting.
[0067] Implementation of the method and system of the present
invention involves performing or completing selected tasks or steps
manually, automatically, or a combination thereof. Moreover,
according to actual instrumentation and equipment of preferred
embodiments of the method and system of the present invention,
several selected steps could be implemented by hardware or by
software on any operating system of any firmware or a combination
thereof. For example, as hardware, selected steps of the invention
could be implemented as a chip or a circuit. As software, selected
steps of the invention could be implemented as a multiplicity of
software instructions being executed by a computer using any
suitable operating system. In any case, selected steps of the
method and system of the invention could be described as being
performed by a data processor, such as a computing platform for
executing a multiplicity of instructions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0069] In the drawings:
[0070] FIGS. 1A-1C illustrate the method of the present invention
for moving a small object from one location to a neighboring
location using only attractive forces;
[0071] FIGS. 2A-2C illustrate the method of the present invention
for moving a small object from one location to a neighboring
location using attractive and repulsive forces;
[0072] FIG. 3 illustrates the method of the present invention for
moving a small object from one location to a non-neighboring
location by a series of successive transfers to a series of
neighboring locations;
[0073] FIG. 4 is a schematic depiction of a location of the present
invention implemented with a trapping enclosure;
[0074] FIG. 5 is a schematic depiction of a hexagonal matrix of
locations of the present invention;
[0075] FIG. 6A is a schematic depiction of a preferred embodiment
of the cell manipulation device of the present invention;
[0076] FIGS. 6B and 6C are schematic depictions of the tip of a
preferred embodiment of a probe tip end of a cell manipulation
probe of the present invention;
[0077] FIG. 6D is a schematic depiction of an alternative
embodiment of a probe tip end of a cell manipulation probe;
[0078] FIG. 7A is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with a single
conduit;
[0079] FIG. 7B is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with a single
conduit and a cell stop;
[0080] FIG. 7C is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with a single
conduit and a bore narrowing;
[0081] FIG. 7D is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with an axial
conduit and one peripheral conduit;
[0082] FIG. 7E is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with two
peripheral conduits;
[0083] FIG. 7F is a diagram depicting a pump system useful for
introducing reagents through a cell manipulation element;
[0084] FIG. 7G is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with a cell
wall penetrating tool;
[0085] FIG. 7H is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with an axial
conduit equipped with a cell wall penetrating tool;
[0086] FIG. 7I is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element with a
retractable cell wall penetrating tool;
[0087] FIG. 8A is a perspective view of a preferred embodiment of a
cell manipulation probe;
[0088] FIG. 8B is a cross section view of a schematic depiction of
a preferred embodiment of a cell manipulation element;
[0089] FIGS. 9A-9C illustrate a method of producing a cell
manipulation element from glass tubes and rods;
[0090] FIGS. 10A-10C are perspective views of modular cell
manipulation elements;
[0091] FIG. 10D is a perspective view of an assembly plate used in
conjunction with modular cell manipulation elements to make a cell
manipulation probe;
[0092] FIGS. 11A-11C illustrate the transfer of a cell from one
cell manipulation element to a neighboring cell manipulation
element;
[0093] FIG. 12 is a schematic depiction of the teachings of the
present invention applied to the on-line treatment of an
organism;
[0094] FIG. 13A is a perspective view illustrating the transfer of
selected cells from a cell manipulation probe to a biochip
processor; and
[0095] FIG. 13B is a perspective view illustrating the transfer of
selected cells from a biochip processor to a cell manipulation
probe.
DETAILED DESCRIPTION OF THE INVENTION
[0096] The present invention is of a method and a device that allow
the isolation and manipulation of individual small objects, such as
and especially living cells. With the appropriate modifications,
some embodiments of present invention allow the practitioner to
isolate, manipulate, observe and study an individual small object
such as a living cell. By manipulate is meant to physically move
but especially to expose to many different and well-defined
stimuli, including but not limited to chemical and physical
stimuli. Using the teachings of the present invention, complex
multi-step experiments can be reproducibly performed on single
individual small objects such as living cells.
[0097] The principles and operation of a device and method of the
present invention may be better understood with reference to the
drawings and accompanying descriptions.
[0098] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the device of the present
invention is not limited in its application to the details of
construction and the arrangement of the components set forth in the
following description or illustrated in the drawings. It is also to
be understood that the method of the invention is not limited in
its application to the details set forth in the following
description or exemplified by the specific embodiments. The
invention is capable of other embodiments or of being practiced or
carried out in various ways. Also, it is to be understood that the
phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.
Method of Moving Small Objects
[0099] A first aspect of the present invention is of a method for
moving a small object from a first location to a second location,
where preferably the relative position of the locations to each
other is fixed.
[0100] By small object is meant an object having dimensions in the
order of less than 10.sup.3 microns, or even less than 10.sup.2
microns, or even less than 101 microns or even less than 10.sup.0
microns. Such objects include cells but also small particles,
crystals, bacteria, viruses, proteins, polymers, macromolecules and
ions.
[0101] The simplest embodiment of the method of the present
invention, depicted in FIG. 1, involves:
[0102] a. applying a first attractive force 100 from a first
location 102 on a small object 104, in order to localize small
object 104 at first location 102 (FIG. 1A); and
[0103] b. thereafter applying a second attractive force 106 from a
second location 108 on small object 104 (FIG. 1B).
[0104] As is clear to one skilled in the art, in such a way small
object 104 is transferred from first location 102 to second
location 108. As is also clear to one skilled in the art, the
proximity and relative spatial orientation of first location 102
and second location 108 must be selected so as to allow efficient
transfer of small object 104.
[0105] To expedite the transfer of small object 104, the intensity
of first attractive force 100 is preferably reduced or eliminated.
Reduction or elimination of first attractive force 100 occurs
before, during or after application of second attractive force 106.
In some embodiments of the method of the present invention the
first attractive force is not only reduced but also reversed to be
a repulsive force. In such a case, the small object is sequentially
or simultaneously repelled from first location 102 and attracted to
second location 108, FIG. 2.
[0106] The method of the present invention is preferably applied to
the moving of some small object from some initial location to some
destination location by the movement of the small object through a
succession of intermediate locations until the small object arrives
at the destination location. Preferably, the position of the
locations relative to each other is fixed. This process is
schematically depicted in FIG. 3 for the movement of small object
104 from an initial location 110 to a destination location 114 by
the movement of small object 104 from initial location 110 through
a succession of three intermediate locations 112a, 112b and 112c
until small object 104 arrives at destination location 114. The
movement of small object 104 between any two locations is analogous
to the movement between first location 102 and second location 108
as depicted in FIG. 1 and FIG. 2 discussed hereinabove.
[0107] When the method of the present invention is applied to a
system where there are more than two locations then any two
locations are related to each other in one of two ways: neighboring
and not-neighboring. Neighboring locations are those locations
where to a small object can be directly transferred in one step.
Non-neighboring locations are those locations where to a small
object can be transferred only via other locations. In FIG. 3,
location 110 and location 112b are neighboring to location 112a.
However location 110 is not-neighboring to location 112b.
[0108] The physical nature of the different locations as well as
the nature of the respective attractive and, if implemented,
repulsive forces depends on the size and nature of the small
objects to be manipulated.
[0109] The attractive and repulsive forces can be physical,
electrical, magnetic or any combination thereof. Electric forces
can include those made by charging a surface, but can also be of
chemical origin such as ionic bonds or hydrogen bonds. Preferably
the attractive and repulsive forces are implemented by a flow of a
physical medium such as a gas or a liquid. When an attractive force
is generated by the flow of a physical medium, then the attractive
force associated with a location is generally implemented by
suction of a fluid through or in the immediate vicinity of that
location. When a repulsive force is generated by the flow of a
physical medium, then the repulsive force associated with a
location is generally implemented by injection of a fluid through
or in the immediate vicinity of that location.
[0110] As stated above, the physical nature of the locations is
determined by the size and nature of the small object to be
manipulated and the nature of the forces applied to manipulate the
object. In some embodiments of the present invention, the locations
are simply areas, for example, on a surface, defined by the
proximity of a respective attractive force. In other embodiments,
the designated locations are dimples or recesses on a surface.
[0111] In preferred embodiments of the present invention,
especially suited for the manipulation of cells, the locations are
trapping enclosures (e.g., chambers or tubes) configured to
substantially physically contain (partially or completely) a small
object, such as a cell. In such preferred embodiments,
schematically depicted in FIG. 4 in cross section, a trapping
enclosure 116 is provided with an open trapping-end 118 through
which a respective attractive or repulsive force is applied and
through which a manipulated small object 120 enters and exits
trapping enclosure 116. It is preferred that the dimensions of open
trapping-end 118 are such that only one small object 120 can enter
trapping enclosure 116 at one time and that once a first small
object 120 enters any other small object, e.g. 122, is blocked from
entering trapping enclosure 116. The geometry of open trapping-end
118 is not generally of significance and can be fashioned in any
shape, for example, square, circular or hexagonal.
[0112] Cells are ordinarily found in a liquid medium. Thus, for
embodiments of the present invention directed to the manipulation
of cells, an attractive force is preferably implemented using a
flow generator 124 operated in a suction mode. Analogously, in such
embodiments a repulsive force is preferably implemented using a
flow generator 124 operated in an injection mode. The inlet/outlet
of flow generator 124 is preferably attached to trapping enclosure
116 in a way so that attractive and repulsive forces act through
open trapping-end 118. In FIG. 4, the inlet/outlet of flow
generator 124 is attached to trapping enclosure 116 through conduit
126 which emerges inside trapping enclosure 116.
[0113] By a flow generator is meant a device or other means to
generate a flow with a desired direction in a fluid. An example of
a flow generator is a pump or plurality of pumps. In some
embodiments a flow generator include valves and the like.
[0114] For embodiments of the present invention directed to the
manipulation of cells, the fluid preferably used is a liquid media,
especially a cell culture media. Many different cell culture media
are known to one skilled in the art. Examples of cell culture media
that are useful in implementing the teachings of the present
invention include but are not limited to Dulbecco's minimal
essential medium, Hank's medium and those listed in U.S. Pat. No.
6,692,961, U.S. Pat. No. 6,689,594, U.S. Pat. No. 6,686,190, U.S.
Pat. No. 6,673,591, U.S. Pat. No. 6,670,184, U.S. Pat. No.
6,670,180, U.S. Pat. No. 6,667,034, U.S. Pat. No. 6,660,501, U.S.
Pat. No. 6,649,408, U.S. Pat. No. 6,642,050, U.S. Pat. No.
6,635,448, U.S. Pat. No. 6,627,426, U.S. Pat. No. 6,610,516, U.S.
Pat. No. 6,593,140, U.S. Pat. No. 6,589,765, U.S. Pat. No.
6,588,586, U.S. Pat. No. 6,569,422, U.S. Pat. No. 6,555,365, U.S.
Pat. No. 6,544,788, U.S. Pat. No. 6,528,286, U.S. Pat. No.
6,511,430, U.S. Pat. No. 6,506,598, U.S. Pat. No. 6,492,163, U.S.
Pat. No. 6,492,148, U.S. Pat. No. 6,489,144, U.S. Pat. No.
6,479,252, U.S. Pat. No. 6,468,788, U.S. Pat. No. 6,465,205, U.S.
Pat. No. 6,465,000, U.S. Pat. No. 6,455,310, U.S. Pat. No.
6,413,746, U.S. Pat. No. 6,413,744, U.S. Pat. No. 6,410,309, U.S.
Pat. No. 6,403,369, U.S. Pat. No. 6,383,810, U.S. Pat. No.
6,378,527, U.S. Pat. No. 6,372,494, U.S. Pat. No. 6,344,354, U.S.
Pat. No. 6,342,384, U.S. Pat. No. 6,338,964, U.S. Pat. No.
6,333,192 and U.S. Pat. No. 6,329,195.
[0115] When manipulating a small object according to the present
invention it is often advantageous to hold the small object at a
given location for a desired length of time. Holding a small object
at a given location can be implemented in many different ways. One
preferred method of holding a small object at a given location is
by the continuous application of an associated attractive force. In
FIG. 4, such a method is implemented by continuously activating
flow generator 124 in suction mode. The use of such a method of
holding the small object in place is generally contingent on the
presence of a "stop", that is a physical impediment to small object
120 being sucked into flow generator 124 by the attractive force.
In FIG. 4 the stop is substantially the small dimension, relative
to the size of small object 120, of conduit 126.
[0116] Another preferred method of holding a small object at a
given location is by relying on the nature of the small object and
the geometry of the location to prevent drifting of the small
object away from the location. In FIG. 4, such a method is
implemented by setting flow generator 124 to inactive mode, a mode
where neither an attractive nor a repulsive force is applied. Due
to the small size of open trapping-end 118 and the lack of currents
inside trapping enclosure 116, small object 120 remains trapped in
the location defined by trapping enclosure 116.
[0117] In one embodiment of the present invention, locations are
disposed in a one-dimensional array, understood to mean that any
location has at most two neighboring locations. Examples of
one-dimensional arrays include linear arrangements of locations,
curved arrangements of locations and circular arrangements of
locations.
[0118] In another embodiment of the present invention, locations
are disposed in a two-dimensional array, understood to mean that at
least one given location has more than two neighboring locations.
Two-dimensional arrays include Y-shaped and X-shaped arrays.
Preferably a two-dimensional array of the present invention is a
matrix arrangement, that is a regular arrangement of a multiplicity
of locations. One preferred matrix arrangement of locations is a
square lattice, where substantially each non-edge location has four
neighboring locations. Another preferred matrix arrangement of
locations is a hexagonal lattice, where substantially each non-edge
location has six neighboring locations. In FIG. 5 a hexagonal
matrix of locations 128 is depicted, each location 128 having a
circular cross section.
[0119] It is important to note that for the sake of simplicity,
arrays of locations are depicted and described herein as being
substantially planar, that is, the locations or the open
trapping-ends of the locations as a group substantially define a
plane. This is done exclusively for the sake of simplicity of
explanation. In some embodiments of the present invention a matrix
of locations implementing the method of the present invention may
be non-planar.
Cell Manipulation Device
[0120] For the manipulation of cells the method of the present
invention is preferably implemented using a cell-manipulation
device of the present invention, the cell-manipulation device
including at least two (but preferably a multiplicity) of
cell-manipulation elements of the present invention, the at least
two (or the multiplicity) of cell-manipulation elements defining a
cell-manipulation probe of the present invention.
[0121] A preferred embodiment and the currently known best mode of
implementing the device of the present invention is described
hereinbelow and with reference, inter alia, to FIG. 6. It is
understood that cell-manipulation device 130 depicted in FIG. 6 and
described hereinbelow is a particular specific device suitable for
manipulating cells in a liquid medium, and described herein
exclusively for exemplary purposes.
[0122] The central component of device 130, a cell-manipulation
probe 132 having a probe-end 134 and a butt end 136 is depicted in
FIG. 6A. Surrounding probe-end 134 is vial 138. Cell manipulation
probe 132 is made up of a multiplicity of bundled cell-manipulation
elements. Functionally associated with each cell-manipulation
element is one, two, three or even more independently controllable
pumps 142. Each pump 142 is connected to a respective
cell-manipulation element through butt end 136 of cell manipulation
probe 132. For clarity, only a limited number of pumps 142 and
connections to cell-manipulation elements are depicted in FIG. 6A.
Controlling the activation of pumps 142 is central control unit
144, substantially a programmable computer with necessary hardware
and software to receive commands from a user input interface 146,
to perform the received commands, for example by performing
necessary calculations or activating a specific pump 142, and to
display information using a user output interface 148. Device 130
is also provided with an observation component 150, such as but not
limited to a CCD camera equipped with a microscope lens.
Observation component 150 in FIG. 6A is functionally associated
with central control unit 144. Observation component 150 receives
commands from central control unit 144 and returns data
corresponding to acquired images, especially of cells manipulated
at probe-end 134 of cell manipulation probe 132. Central control
unit 144 is configured to display data received from observation
component 150 and display the data in user understandable form
through user output interface 148. It is preferred that central
control unit 144 be configured with image processing capabilities
so as to "interpret" video data received from observation component
150 and react to the interpreted data.
[0123] In FIG. 6B, a close-up of probe-end 134 of cell-manipulation
probe 132 without vial 138 is depicted. A multiplicity of hexagonal
trapping enclosures 140, each being a part of a cell-manipulation
element, are depicted with a cell 142 trapped therein. Trapping
enclosures 140 are arrayed in a matrix 145. A semi-circular
isolation inlet 146 of an isolation element and a semi-circular
waste inlet 148 of a waste element are positioned at the
circumference of matrix 145. In the center of matrix 145 is a
multi-cell reaction enclosure 151 of a multi-cell reaction
element.
[0124] In FIG. 6C, probe-end 134 of cell manipulation probe 132
with vial 138 is depicted close-up. Emerging into vial 138 are
sample inlet 152, fluid inlet 154 and vial reagent inlet 156. Each
such inlet is functionally associated with a respective pump 158,
160 and 162 to allow introduction of samples, fluid or reagents,
respectively, onto probe-end 134. Pumps 158, 160 and 162 are
independently controllable by central control unit 144.
Cell-Manipulation Elements
[0125] As stated hereinabove, cell-manipulation probe 132 is
substantially made up of a multiplicity of individual
cell-manipulation elements. There are many variations of
cell-manipulation elements, some of which are discussed
hereinbelow.
Types of Cell-Manipulation Elements
[0126] One simple cell manipulation element preferred in
implementing a device of the present invention is a dimple on a
surface, where at the bottom of the dimple is the inlet of an
functionally associated flow generator having two modes: a suction
mode (to generate an attractive force) and an inactive mode. When
the flow generator is set to suction mode, a cell is attracted and
held at the associated dimple by the produced suction. When the
pump is set to inactive mode, the held cell may drift away. If a
pump functionally associated with a neighboring dimple is set to
suction mode, the cell drifts towards and is held in that
neighboring dimple in accordance with the method of the present
invention. Although such a cell manipulation element is simple to
produce and operate, and a cell-manipulation probe made of an array
of such cell manipulation elements effectively implements the
method of the present invention, the utility of such a
cell-manipulation probe is limited as it is difficult to expose
only selected cells to stimuli such as reagents, vide infra.
[0127] For embodiments of the present invention directed to the
manipulation of cells, it is generally preferred that a cell
manipulation element have a trapping enclosure with an open
trapping end as depicted in FIG. 4. The trapping enclosure is
configured so as to substantially physically contain (partially or
completely) a cell held therein while the open trapping end
provides access of cells into and out of the trapping enclosure. In
such a way a cell in the trapping enclosure is isolated, allowing
selective exposure of cells to stimuli such as reagents. The
inlet/outlet of a flow generator associated with a cell
manipulation element having a trapping enclosure advantageously
emerges within the trapping enclosure. In some embodiments such a
flow generator has only two modes: a suction mode and an inactive
mode. In a preferred embodiment, such a trapping enclosure is
configured or shaped so that when the flow generator applies no
force (e.g. is set to inactive mode), a held cell is retained
within the trapping enclosure. When the trapping enclosure is so
configures, an associated flow generator preferably has at least
three modes: a suction mode (to generate an attractive force), an
injection mode (to generate a repulsive force) and an inactive
mode. When the flow generator is set to suction mode, fluid is
drawn from a trapping enclosure through a respective conduit
generating an attractive force to pull a nearby cell into the
trapping enclosure. When the flow generator is set to inactive
mode, a held cell remains within the trapping enclosure. When the
flow generator is set to injection mode, fluid is injected into a
trapping enclosure through a respective conduit generating a
repulsive force to eject a held cell from the trapping
enclosure.
[0128] It is generally preferred that a trapping enclosure and an
open trapping end of a cell manipulation element have dimensions
close to those of the cell to be held and manipulated. As is known
to one skilled in the art, cells of various sizes exist. Plant
cells are typically large, having a diameter of between 10.sup.1 to
10.sup.2 microns. Animal cells typically have a diameter of between
10.sup.-1 to 10.sup.1 microns. Bacteria are typically small, having
a diameter of between 10.sup.-1 to 10.sup.-2 microns. It is
therefore preferred that the dimensions of the open trapping-ends
of the trapping enclosure be determined by the size of cells that
are to be studied with a specific device of the present
invention.
Cell Manipulation Elements Associated with a Single Conduit
Pump
[0129] A simple cell manipulation element 164 implementing a
trapping enclosure is schematically depicted in FIG. 7A. Cell
manipulation element 164 is substantially a tube 166 functionally
associated with a conduit pump (not depicted), the conduit pump
operable in a suction mode, an injection mode and an inactive mode.
In cell-manipulation element 164 the end of tube 166 serves as an
open trapping-end 170 and the bore of tube 166 serves as a trapping
enclosure 140. The bore of tube 166 also serves as a conduit 174
for transporting fluid from the functionally associated conduit
pump to attract and repel a cell in accordance with the teachings
of the present invention.
[0130] It is often preferred that a cell manipulation element have
a cell stop, a physical obstruction limiting the extent of
penetration of a cell inside a cell manipulation element. Depending
on the specific embodiment, a cell stop is a narrowing, a
protrusion or other physical structural feature that limits the
distance into an functionally associated trapping enclosure that a
cell can be drawn.
[0131] A preferred cell manipulation element 176 integrating a cell
stop is depicted in FIG. 7B. Cell manipulation element 176 is
similar to cell manipulation element 164 except that cell
manipulation element 176 is also provided with a cell stop 178.
Cell stop 178 obstructs the bore of tube 166 so that a cell
entering through open trapping-end 170 penetrates until making
contact with cell stop 178. Trapping enclosure 140 of cell
manipulation element 176 is defined by the inside surface of tube
166 and cell stop 178. The volume between cell stop 178 and tube
166 serves as a single conduit 174 for transporting fluid from a
functionally associated conduit pump to attract and repel a cell in
accordance with the teachings of the present invention. It is
important to note that in some embodiments conduit 174 has an
annular cross section. In other embodiments conduit 174 is made of
a multiplicity of individual fluid paths peripherally arrayed about
cell stop 178. In such an embodiment, all the individual fluid
paths preferably transport fluid from a single conduit pump.
[0132] An additional cell manipulation element 180 integrating a
cell stop is depicted in FIG. 7C. In cell manipulation element 180
the function of a cell stop is performed by a narrowing 181 of the
bore of tube 166 to an extent that a cell entering through open
trapping-end 170 cannot penetrate beyond narrowing 181. Thus,
trapping enclosure 140 of cell manipulation element 180 is defined
by the inside surface of tube 166 and narrowing 181 in the bore of
tube 166. As in cell manipulation element 164, the bore of tube 166
serves as a conduit 174 for fluid from a functionally associated
conduit pump.
[0133] Cell manipulation elements 164, 176 and 180 are all operated
in a substantially similar way. Central control unit 144 sets a
functionally associated conduit pump to suction mode, producing an
attractive force through conduit 174 to attract cells in the
vicinity of an open trapping-end 170 to enter cell manipulation
element 164, 176 or 180 through an open trapping-end 170 and to be
trapped in a trapping enclosure 140. When cell manipulation element
164 is used, central control unit 144 sets a functionally
associated conduit pump to inactive mode when a cell enters open
trapping-end 170 to prevent penetration of the cell too far into
the bore of tube 166 and consequent loss of the cell. It is thus
generally preferred to use a cell manipulation element such as 164
under observation of an observation component 150. In contrast, a
cell entering open trapping-end 170 of a cell manipulation element
176 or 180 makes contact with a functionally associated cell stop
178 or narrowing 181, respectively, and thus remains held in
trapping enclosure 140.
[0134] When it is desired to eject a cell from a trapping enclosure
140 of a cell manipulation element 164, 176 or 180, central control
unit 144 sets a respective functionally associated conduit pump to
injection mode. Fluid is forced through conduit 174 pushing a held
cell outwards from trapping enclosure 140 through open trapping end
170.
Cell Manipulation Elements Functionally Associated with Multiple
Conduit Pumps
[0135] To increase the flexibility of the types of cell
manipulations that can be performed using a device of the present
invention, it is often advantageous to use a cell manipulation
element having more than one conduit and functionally associated
with more than one independently controllable conduit pump.
[0136] Cell manipulation element 182, depicted in FIG. 7D, has more
than one conduit and is functionally associated with more than one
independently controllable conduit pump. Cell manipulation element
182 is related to cell manipulation element 176 in that a trapping
enclosure 140 is defined by the inside surface of a tube 166 and by
a cell stop 184. Further, the volume between tube 166 and cell stop
184 serves as a first peripheral conduit 174 for fluid from a
functionally associated first conduit pump. Cell manipulation
element 182 is characterized, however, in that cell stop 184 is
substantially a hollow tube. The bore of cell stop 184 serves as a
second axial fluid conduit 186 transporting fluid from an
independently controlled and activated second conduit pump.
[0137] An additional preferred cell manipulation element
functionally associated with more than one independently
controllable conduit pump is 188 depicted in FIG. 7E. Cell
manipulation element 188 is related to cell manipulation element
176 in that a trapping enclosure 140 is defined by the inside
surface of a tube 166 and by a cell stop 178. However, in cell
manipulation element 178, the volume between tube 166 and cell stop
178 does not have an annular cross section but is rather made up of
at least two distinct conduits found at the periphery of tube 106,
a first peripheral conduit 174 and a second peripheral conduit 186.
First peripheral conduit 174 is functionally associated with a
first conduit pump. Second peripheral conduit 186 is functionally
associated with an independently controlable second conduit
pump.
[0138] It is important to note that variants of cell manipulation
element 188 are provided with more than two (e.g. three, four,
five, six or even more) conduits (especially peripheral conduits)
each such conduit associated with an individually controllable
conduit pump.
[0139] For loading a cell into a cell manipulation element
functionally associated with two independently controllable pumps,
a functionally associated first conduit pump and/or second conduit
pump are set to suction mode by central control unit 144. If only
one associated conduit pump is used, the other associated conduit
pump is set to inactive mode. Analogously, ejection of a cell from
trapping enclosure 140 of cell manipulation element 182 is
performed when a functionally associated first conduit pump and/or
second conduit pump are set to injection mode by central control
unit 144. If only one associated conduit pump is used, the other
associated conduit pump is set to inactive mode.
[0140] A useful function that can be implemented using a cell
manipulation element functionally associated with two or more
independently controllable conduit pumps is washing of a cell held
in a respective trapping enclosure 140. For a cell manipulation
element 188, central control unit 144 sets one functionally
associated conduit pump to suction mode and the other functionally
associated conduit pump to injection mode. Liquid flows from one
conduit to another, thus washing a cell held in trapping enclosure
140. For a cell manipulation element 182, central control unit 144
sets a first conduit pump functionally associated with peripheral
conduit 174 to injection mode and sets a second conduit pump
functionally associated with axial conduit 186 to suction mode.
Liquid flows from one conduit to the other, thus washing a cell
held in trapping enclosure 140.
[0141] A useful function that can be performed using cell
manipulation element 182 is suspending a cell held in trapping
enclosure 140 at a distance from open trapping-end 170. To perform
the suspension function, central control unit 144 sets the conduit
pump functionally associated with axial conduit 186 to injection
mode while setting the conduit pump functionally associated with
peripheral conduit 174 to suction mode. The resulting Venturi
effect suspends a cell originally trapped in trapping enclosure 140
above open trapping-end 170. The suspension function allows a cell
to be temporarily removed from trapping enclosure 140 to allow
clear, unimpeded examination of the cell from many directions, a
challenging task when a cell is contained within trapping enclosure
140. The suspension function as described hereinabove may be
generally performed with a properly configured cell manipulation
element having at least one axial conduit and at least one
peripheral conduit. If there is only one peripheral conduit, that
peripheral conduit is preferably substantially annular. If there is
more than one peripheral conduit, the peripheral conduits are
preferably arrayed symmetrically about the axial conduit.
[0142] An additional useful function that can be implemented using
a cell manipulation element functionally associated with two or
more independently controllable conduit pumps, such as 182 or 188,
is selective exposure of a cell held in trapping enclosure 140 to a
reagent. The function of selectively exposing a cell to a reagent
will be discussed herein with reference to a cell manipulation
element 188. In one embodiment, exposure is performed by providing
one of the two conduit pumps functionally associated with cell
manipulation element 188 with a reagent. When it is desired to
selectively expose a cell to the reagent, a process analogous to
washing a cell as described above is followed. The associated
conduit pump provided with reagent is set to injection mode
bringing reagent into trapping enclosure 140 and thus to contact a
cell held therein. In parallel the second associated conduit pump
is set to suction mode, drawing away reagent and preventing leakage
of reagent out through a open trapping-end 170. A disadvantage of
such an implementation is that a conduit pump provided with reagent
cannot be used for performing other functions.
[0143] Selective exposure of a cell can also be achieved in a cell
manipulation element provided with three conduits each functionally
associated with an independently controllable conduit pump, where
one conduit is dedicated to providing reagent and the other two
conduits only for performing other functions.
[0144] In a preferred embodiment for implementing selective
exposure of cells to a reagent, a cell manipulation element is
functionally associated with three pumps as depicted in FIG. 7F. In
FIG. 7F is depicted a cell manipulation element 188 where conduit
174 is functionally associated with a first conduit pump 190 and
conduit 186 is functionally associated with a second conduit pump
192. On the fluid-line connecting conduit 174 with first conduit
pump 190 is reagent injection pump 194. Similarly, to the cell
washing function discussed hereinabove, when it is desired to
expose a cell to a reagent, central control unit 144 sets first
conduit pump 190 to injection mode and second conduit pump 192 to
suction mode. Further, central control unit 144 sets reagent
injection pump 194 to injection mode, causing reagent from a
container 196 to enter the flow of first conduit pump 190 towards
conduit 174. The reagent is transported through conduit 174,
exposing a cell held in trapping enclosure 140 to the reagent.
Reagent is then pumped away through conduit 186 by the action of
second conduit pump 192, drawing away reagent and preventing
leakage of reagent out through a open trapping-end 170.
Cell Manipulation Elements Having a Tool for Penetrating a Cell
Wall
[0145] It is often advantageous to be able to introduce one or more
reagents directly into a cell or to extract a sample from inside a
cell. To this end, in a preferred embodiment of the present
invention, a cell manipulation element is provided with a tool
configured to penetrate a cell wall. Such a tool is, for example, a
pointed solid object, for example from glass or metal. Preferably,
such a tool is disposed inside the trapping enclosure of the cell
manipulation element in such a way that when an attractive force
according to the present invention is applied, the cell is pulled
against the tool and the cell wall consequently penetrated.
[0146] In FIG. 7G a cell manipulation element 198 analogous to cell
manipulation element 188 is depicted, having a cell wall
penetrating tool 200. Cell wall penetrating tool 200 is a stainless
steel wire inside cell stop 178. To penetrate the wall of a cell,
the cell is brought into trapping enclosure 140. Conduit pumps
functionally associated with conduits 174 and 186 are set to
suction mode. The resulting attractive force pulls the cell onto
cell wall penetrating tool 200, thus penetrating the cell wall.
Post-penetrating cell release is accomplished by setting the
functionally associated conduit pumps to injection mode so that the
resulting liquid flow through conduits 174 and 186 pushes the
penetrated cell off of cell wall penetrating tool 200.
[0147] Advantageous is that once a cell wall is penetrated, a
reagent is injected into the penetrated cell or the contents of the
penetrated cell are sampled. In a preferred embodiment that a cell
wall penetrating tool has a conduit functionally associated with an
independently controllable pump for injecting reagents or sampling
cell contents through the conduit.
[0148] In FIG. 7H a cell manipulation element 202 analogous to cell
manipulation element 182 is depicted, having a cell wall
penetrating tool 204. Cell wall penetrating tool 204 is
substantially a sharpened tip of hollow cell stop 184. To penetrate
the wall of a cell, the cell is brought into trapping enclosure
140. The conduit pumps functionally associated with peripheral
conduit 174 are set to suction mode so that the resulting liquid
flow pulls the cell onto cell wall penetrating tool 204,
penetrating the cell wall. If it is desired to extract a sample
from inside the cell, the conduit pump functionally associated with
axial conduit 186 is set to suction mode. If it is desired to
inject a reagent into the cell, the conduit pump functionally
associated with axial conduit 186 is set to injection mode. As
described for cell manipulation element 198, post-penetrating cell
release is accomplished by setting conduit pumps functionally
associated with peripheral conduit 174 to injection mode so that
the resulting liquid flow pushes the penetrated cell off from cell
wall penetrating tool 204.
[0149] An alternative method for releasing a penetrated cell from a
cell wall penetrating tool is by providing a cell manipulation
element 206 with a retractable cell wall penetrating tool 208 as
depicted in FIG. 7I. Cell manipulation element 206 is similar to
cell manipulation element 182 excepting that a cell wall
penetrating tool 208, a stainless steel wire, is threaded through
bore 186.To penetrate the wall of a cell, the cell is brought into
trapping enclosure 140. Pumps functionally associated with conduits
174 and 186 are set to suction mode. The resulting attractive force
pulls the cell onto cell wall penetrating tool 208, thus
penetrating the cell wall. Post-penetrating cell release is
accomplished by pulling cell wall penetrating tool 208 outwards
from the penetrated cell through bore 186.
Multi-Cell Reaction Element
[0150] A multi-cell reaction element, of which only a multi-cell
reaction enclosure 150 is depicted in FIG. 6B, is a substantially a
cell-manipulation element having an open trapping-end and a
multi-cell reaction enclosure 150 both large enough to accommodate
at least two cells rather than only one cell like a standard
cell-manipulation element as described hereinabove. A multi-cell
reaction element can be substantially of any of the types of
elements discussed hereinabove (especially as related to the number
and arrangement of conduits) and is functionally associated with at
least one independently controllable conduit pump.
[0151] A multi-cell reaction element provides a venue to isolate
two or more cells in one place to enable interaction of the two or
more cells. Operation of a multi-cell reaction element is analogous
to the operation of cell manipulation elements as described above.
As depicted in FIG. 6B, at least two cells 142, each isolated in a
separate trapping enclosure 140 are moved according to the method
of the present invention to trapping enclosures 140 neighboring
multi-cell reaction enclosure 150. The at least two cells 142 are
expelled from respective trapping enclosures 140 while a conduit
pump functionally associated with the multi-cell reaction element
is set to suction mode. The resulting forces draw the expelled
cells into multi-cell reaction enclosure 150. Inside multi-cell
reaction enclosure 150 the at least two cells 142 interact. If
desired, reagents are added as described hereinabove and
hereinbelow. When the cell interaction is completed the at least
two cells 142 are expelled from multi-cell reaction enclosure 150.
The at least two cells 142 can be discarded or reisolated in
trapping enclosures 140 in the usual way.
[0152] Importantly, in a preferred embodiment a cell manipulation
probe of the present invention is provided with more than one
multi-cell reaction element.
Sample Introduction Element
[0153] In FIG. 6D, in the center of matrix 145 emerges the outlet
of sample introduction element 163. Sample introduction element is
substantially a tube functionally associated with an inlet pump
(not depicted). Samples containing cells that are to be manipulated
using the device of the present invention are introduced through
sample introduction element 163.
Isolation Element
[0154] An isolation element, of which only a substantially
semi-circular isolation inlet 146 is depicted in FIG. 6B, is
substantially a multi-cell reaction element with significant
structural differences arising from the different uses for which
these two elements are intended. An isolation element is primarily
intended to provide a location where a user of a device of the
present invention concentrates a relatively large number of cells
without necessarily intending to later manipulate the cells as
individuals. For example, certain cells can be sorted from a large
group of cells, and only the certain cells are selected and
transferred to an isolation enclosure of the isolation element.
From the isolation enclosure the certain cells are removed, either
in accordance with the method of the present invention or in some
other manner. Therefore, an isolation enclosure that is part of an
isolation element is generally large enough to accommodate a
relatively large volume of fluid and many cells. At a minimum,
isolation element is functionally associated with an isolation pump
configured to operate in a suction mode. When the isolation pump is
operated, cells are drawn into the isolation enclosure of the
isolation element. In some embodiments, an isolation element is
functionally associated with an isolation pump configured to
operate also in an injection mode. When the isolation pump is
operated in injection mode, cells trapped in the isolation
enclosure are expelled back to the vicinity of probe tip 134.
Preferably, the isolation enclosure of an isolation element is
provided with a diaphragm or other valve-like structural feature. A
user can, using a needle-equipped syringe (or other appropriate
device), draw out cells and fluids isolated in the isolation
enclosure.
[0155] Importantly, in a preferred embodiment a cell manipulation
probe of the present invention is provided with more than one
isolation element.
Waste Element
[0156] A waste element, of which only a substantially semi-circular
waste inlet 148 is depicted in FIG. 6B, is substantially a tubular
element functionally associated with a waste pump operable in
suction mode. The function of waste element is to remove undesired
or not needed substances from the proximity of probe-end 134 of
cell-manipulation probe 132, and especially from matrix 145. When a
functionally associated waste pump is activated, cells, fluid, cell
debris and the like are drawn through waste inlet 148 of the waste
element and discarded.
Cell-Manipulation Probe
[0157] A cell-manipulation probe of the present invention is
composed of at least two but preferably a multiplicity of
individual cell-manipulation elements as described above. The
cell-manipulation elements are arranged so that the open
trapping-ends of the cell-manipulation elements are in functional
proximity of each other, that is at a distance and orientation so
that cells can be transferred from a first cell-manipulation
element to a neighboring cell-manipulation element according to the
method of the present invention. Preferably but not necessarily a
cell-manipulation probe is also provided with a sample introduction
element, waste element, an isolation element and/or a multi-cell
reaction element.
[0158] In some embodiments, a cell-manipulation probe may be
homogenous, that is that all the component cell-manipulation
elements are substantially the same. In other embodiments, a
cell-manipulation probe is heterogeneous, that is the
cell-manipulation probe includes a variety of different
cell-manipulation elements. Differences between the
cell-manipulation elements making up a heterogeneous cell
manipulation probe potentially include the geometry of the trapping
enclosures (especially size of the trapping enclosure or size of
the open trapping-ends), the number of conduits and the
arrangements of conduits. A cell-manipulation probe potentially
includes some or all of the types of cell manipulation elements
discussed hereinabove, but potentially types of cell-manipulation
elements not discussed herein but may arise are also included. The
utility of a heterogeneous cell-manipulation probe is discussed
hereinbelow.
[0159] In a preferred embodiment, a cell manipulation probe is a
bundle of cell manipulation elements. In FIG. 8A a cell
manipulation probe 132 is depicted. In FIG. 8B a component cell
manipulation element 210 similar to cell manipulation element 176
is depicted in cross section. As seen in FIG. 6A and in FIG. 8A a
cell manipulation probe 132 has a probe-end 134 and a butt end 136.
As depicted in FIG. 8A, generally speaking, at butt end 136 of cell
manipulation probe 132 the ends of the individual cell manipulation
elements 210 are relatively large (.about.10.sup.3 microns, 1000 to
2000 microns) so as to allow simple maintenance and attachment of
conduits and cell manipulation elements to functionally associated
pumps and the like. Generally speaking, at probe-end 134 of cell
manipulation probe 132 the ends of individual cell manipulation
elements 210 make up a matrix 145 of respective open trapping-ends
140 and have dimensions determined by the uses and size of cells to
be manipulated, which are generally significantly smaller in the
micron scale, FIG. 6B. For example, the size of open trapping ends
for the manipulation of cells is in the order of between 10.sup.-1
to 10.sup.2 microns. Therefore a typical cell-manipulation element
210 and a typical cell manipulation probe 132 are both of a
gradually decreasing dimension from butt-end 136 towards probe-end
134, as depicted in FIG. 8.
Observation Component
[0160] Although the device of the present invention can be used to
manipulate cells without actually observing any individual cell
manipulated, it is generally preferable to observe the cells.
Observation is useful, for example, for selecting cells for
manipulation, for morphological study, for direct observation of
biological processes and to confirm that desired manipulations are
performed as desired. It is therefore preferable that a device 130
of the present invention be functionally provided with an
observation component 150, as depicted in FIG. 6A.
[0161] Many different types of observation components can be
usefully coupled to a device of the present invention, but
preferably an observation component is an optical inspection
device. Optical inspection devices such as optical or CCD
microscopes having a magnification appropriate for viewing living
cells in sizes in the order of 10.sup.-1 microns are known and are
commercially available.
[0162] As is known to one skilled in the art, an optical or CCD
microscope is most effective for the morphological and morphometric
characterization of a studied object. It is therefore advantageous
to provide a device of the present invention, such as device 130,
with an observation component suited for the detection of
fluorescence. It is known to one skilled in the art that the
measurement of vital fluorescence parameters (fluorescence
intensity, polarization, energy transfer) provides important
information about a living cell.
Central Control Unit
[0163] To allow performance of complex experiments it is preferred
that a device of the present invention such as 130 be provided with
a central control unit 144 as depicted in FIG. 6A. A typical
central control unit such as 144 receives commands from a user
through a user input interface 146 and activates pumps 142,
observation component 150 and other components as necessary. As is
clear to one skilled in the art, a central control unit 144 can
advantageously be implemented using a programmable computer.
[0164] As is discussed herein, central control unit 144 is also
used to determine the exact sequence of activation of pumps 142 and
other components of a device 130 of the present invention to trap,
move, observe and otherwise manipulate cells using a
cell-manipulation probe 132.
[0165] In a preferred embodiment, a central control unit 144 is
used also to store a record of the experiments and conditions to
which each cell was exposed, vide infra.
[0166] Although identification, examination and determination of
various states of a cell can be performed by a human operator
looking at video output produced by an observation component of a
device of the present invention, it is preferred that some or all
of these functions be performed by a central control unit.
Automated image analysis useful in identifying locations of cells
and various morphologies is well-known and can be implemented in
the context of a device of the present invention using a central
control unit by one skilled in the art of image analysis.
[0167] Many other functions required from or possible with a
central control unit are known or obvious and will not be listed or
discussed herein.
Construction of a Cell Manipulation Device
[0168] There are many technologies available allowing one skilled
in the art to construct a cell manipulation probe of the present
invention.
[0169] One preferred method for the construction of a
cell-manipulation probe of the present invention is free-form
manufacturing (FFM) using ceramic powders. As is known to one
skilled in the art, in free-form manufacturing a part, such as a
cell-manipulation probe, is built "from the ground up" by
sequentially dispensing layers of ceramic powder one on top of the
other, see for example, U.S. Pat. No. 6,376,148; U.S. Pat. No.
6,238,614; U.S. Pat. No. 6,228,437; U.S. Pat. No. 6,066,285; U.S.
Pat. No. 6,117,612; U.S. Pat. No. 6,046,426; U.S. Pat. No.
5,059,266; U.S. Pat. No. 5,204,055 or U.S. Pat. No. 6,206,672. A
review of the state of the art of FFM can be found in U.S. Pat. No.
6,376,148, which is incorporated herein by reference for all
purposes as if fully set forth herein. It is important to note that
when a ceramic powder is used it is generally preferable to use a
ceramic powder with as small a diameter as possible, for example 10
nanometer diameter ceramic powders. Since the use of free-form
manufacture methods to construct a complex structure such as a
cell-manipulation probe of the present invention is known to one
skilled in the art, this is not further discussed herein.
[0170] A cell manipulation probe, such as cell manipulation probe
132 can be purchased from commercial suppliers, for example from
TEGS Ltd. (Saratov, Russia).
[0171] A preferred method for the construction of a
cell-manipulation probe, such as cell manipulation probe 132, is
through the bundling of a multiplicity of glass tubes and rods.
Generally speaking, the preferred method involves three steps. In a
first step, glass rods and tubes are bundled together in an
appropriate way to assemble an incipient cell-manipulation element
or, preferably, an incipient cell-manipulation probe. The diameter
of the tubes and rods is chosen so that subsequent attachment of
pumps and the like to the butt end of the cell-manipulation probe
is simple. In a second step, glass rods are fused together. In a
third step, one end of the bundle of glass rods and tubes is drawn
in the usual manner known to one skilled in the art of
glass-working so as to form the probe-end of the cell manipulation
probe. In such a way, the size of the probe-end of the
cell-manipulation probe is reduced without blocking channels and
conduits, producing a cell-manipulation probe as depicted in FIG.
8A.
[0172] The method of use of glass tubes and rods in constructing an
individual cell-manipulation element is described in further detail
with reference to FIG. 9. Extension of the method of arranging
glass tubes and rods to the construction of a cell-manipulation
probe involves the construction of a multiplicity of
cell-manipulation elements and is clear to one skilled in the art
upon study of the disclosure herein.
[0173] FIG. 9A is a top view of a bundle of seven glass rods 212a,
212b, 212c, 212d, 212e, 212f and 212g used in the assembly of a
cell manipulation element whereas FIG. 9B is a vertical cross
section of the bundle through the plane A-A. The bundle of seven
glass rods 212 can be considered to be an incipient
cell-manipulation element 213. It is seen in FIG. 9B that the end
of central glass rod, 212g is lower than the end of glass rods 212e
and 212c. In fact, the ends of all six glass rods 212a through 212f
are at the same height. The volume in incipient cell manipulation
element 213 defined by the sides of glass rods 212a through 212f
and the top end of recessed glass rod 212g corresponds to the
trapping enclosure of the ultimately fashioned cell manipulation
element. The top end of recessed glass rod 212g corresponds to the
cell stop of the ultimately fashioned cell-manipulation element.
The interstitial volumes between seven glass rods 212 correspond to
six fluid passages that become the conduit or conduits in the
ultimately fashioned cell-manipulation element.
[0174] The use of seven glass rods 212 as depicted in FIGS. 9A and
9B and as described hereinabove allows the construction of a
cell-manipulation element such as 176 of FIG. 7B, 188 of FIG. 7E,
198 of 7G and 206 of 71, although for the construction of cell
manipulation elements such as 198, a glass rod having a stainless
steel wire through the central axis may replace glass rod 212g. It
is important to note that generally the differentiation between a
single conduit cell-manipulation element such as 176 and a multiple
conduit cell-manipulation element such as 188, 198 or 206 is not
necessarily apparent in the incipient cell-manipulation element
213. Rather, after the incipient cell-manipulation element (or
incipient cell-manipulation probe) is drawn, the exact nature of
the ultimately fashioned cell-manipulation element is determined by
how functionally associated conduit pumps are attached to the
passages. If all six passages are connected to a single conduit
pump, then a cell-manipulation element having one conduit such as
188 is formed. If two adjacent liquid passages are connected to one
conduit pump, whereas the opposing pair of liquid passages is
connected to a second conduit pump then a cell manipulation element
having two peripheral conduits such as 188, 198 or 206 is
formed.
[0175] In FIG. 9C a method of construction for a cell manipulation
elements such as 182 of FIG. 7D, 202 of 7H or 206 of 7I is
depicted. Analogously to the method described immediately
hereinabove and depicted in FIG. 9A and FIG. 9B, six glass rods
212a, 212b, 212c, 212d, 212e and 212f are arrayed about a glass
tube 214. Since the end of glass tube 214 is lower than the ends of
glass rods 212, the volume defined by the sides of glass rods 212a
through 212f and the top end of recessed glass tube 214 corresponds
to the trapping enclosure of the ultimately fashioned cell
manipulation element. The top end of recessed glass tube 214
corresponds to the cell stop of the ultimately fashioned
cell-manipulation element. The interstitial volumes between six
glass rods 212 and recessed glass tube 214 correspond to six
passages that potentially become conduits in the ultimately
fashioned cell-manipulation element. The hollow bore of recessed
glass tube 214 corresponds to the axial conduit in the ultimately
fashioned cell-manipulation element. To fashion a cell wall
penetrating tool 204 of a cell manipulation element 202, the end of
recessed glass tube 214 is sharpened.
[0176] It is clear to one skilled in the art upon studying the
description herein, that in order to fashion an incipient
cell-manipulation probe instead of an incipient cell manipulation
probe, more than seven glass rods and tubes are bundled together.
It is also clear to one skilled in the art upon reading the
description herein that other types of cell manipulating elements
or other elements for constructing a cell manipulating probe such
as a waste element or an isolation element can be produced in an
analogous fashion to that described hereinabove.
[0177] As stated above, once chosen the glass rods and tubes making
up an incipient cell-manipulation element or incipient cell
manipulation probe are bundled together in an appropriate fashion,
the rods and tubes are fused together. Thereafter, the appropriate
end of the incipient cell-manipulation element or incipient cell
manipulation probe is drawn out into the shape depicted in FIG. 8A
to make a cell manipulation element or cell manipulation probe,
respectively.
[0178] In an alternative embodiment, modular cell manipulation
elements are made and assembled to make a cell manipulation probe.
In FIG. 10 are depicted examples of modular cell manipulation
elements.
[0179] In FIG. 10A is depicted one embodiment of a simple modular
cell manipulation element 216 with two conduits, 218 and 220. The
horizontal cross-section of cell manipulation element 216 is
substantially square. It is important to note that other
embodiments of similar cell manipulation elements potentially have
non-square cross-sections. Conduits 218 and 220 function
analogously to conduits described hereinabove. Illustrative but
non-limiting dimension of cell manipulation element 216 are a
height of 1000 micron, a width of 20 micron and a depth of 20
micron. The top of cell manipulation element 216 is recessed,
creating a well-like enclosure, 222 serving as a dimple or trapping
enclosure as described hereinabove. An illustrative but
non-limiting depth of well-like enclosure 222 is 10 micron. Other
types of elements, especially cell manipulation elements, can be
conceived for use together with modular cell manipulation elements
such as 216. Examples of such other types of elements include a
modular cell manipulation element 224 with three conduits 218, 220,
and 226, depicted in FIG. 10B and a modular cell manipulation
element 228 with two conduits 218 and 220 and a cell wall
penetrating tool 230, depicted in FIG. 10C.
[0180] Assembly of a cell manipulation probe from modular cell
manipulation elements such as 216, 224 and 228 is done by providing
an assembly plate 232, depicted in FIG. 10D. Assembly plate 232 is
a monolithic structure having a multiplicity of pin connector
triplets 234, which fit into the bottom openings of conduits 218,
220 and 226 or a conduit of cell wall piecing tool 230 of modular
cell manipulation elements 216, 224 and 228. At the bottom of
assembly plate 224, each pin connector 226 is connected to fluid
lines (not depicted), which connect to functionally associated
conduit pumps (not depicted). Thus a multiplicity of modular cell
manipulation elements 216 can be disposed in exact juxtaposition on
assembly plate 224, thus providing functionally associated conduit
pumps to cause fluid to flow through conduits 218 and 220.
[0181] The various components necessary for implementing a cell
manipulation probe using modular cell manipulation elements,
including modular cell manipulation elements can be purchased from
commercial suppliers, for example from TEGS Ltd. (Saratov,
Russia).
[0182] Once a cell-manipulation probe is constructed, the assembly
of a device of the present invention from commercially available
components is well within the ability of one skilled in the art
upon study of the description herein.
[0183] Suitable computers, software and related peripheral
equipment for implementing a central control unit are available
from International Business Machines Corporation (Armonk, N.Y.,
USA). Any required unique or dedicated software can be written by
one skilled in the art of computer programming.
[0184] Suitable CCD microscopes for implementation of an
observation component are available, for example, from Hirox Co
Ltd. (Suginami-Ku, Tokyo, Japan).
[0185] Flow-generators and peripheral equipment suitable for
implementing the present invention are available from, for example,
GeSIM (Grosserkmannsdorf, Germany) or Fluidigm Corporation (San
Francisco, Calif., USA).
Steps Used in Building Experiments
[0186] A device such as 130 depicted in FIG. 6 designed for
manipulating individual cells found in a liquid medium (especially
a cell culture medium) and allows performance of many novel and
useful experiments. Experiments are done by performing a series of
one or more steps (also termed manipulations or functions). A few
standard steps that are useful building blocks for various
experiments are described in detail hereinbelow with reference to
device 130 as depicted in FIG. 6 and especially with reference to
FIG. 6B. Unless otherwise stated, for the examples below it is
assumed that all cell manipulation elements are similar to cell
manipulation element 182 depicted in FIG. 7D with a reagent inlet
system such as depicted in FIG. 7F.
[0187] Useful steps, manipulations and functions include loading
and isolating of a cell in a trapping enclosure, washing a cell,
suspending a cell for observation, penetrating a cell wall (e.g.
for removing a sample or inoculation with a reagent), exposure of a
cell to a reagent, allowing a cell to interact with other cells,
select a cell, sort cells and separate types of cells from
others.
Cell Loading and Isolating
[0188] Generally but not necessarily, a first step in an experiment
is the loading of cells into trapping enclosures 140 of device
130.
[0189] Sample inlet pump 158 receives a command from central
control unit 144 to operate, loading a sample of cell-containing
liquid through sample inlet 152 onto matrix 145 on probe-end 134 of
cell manipulation probe 132, FIG. 6C. In an alternate embodiment
using the variant of cell manipulation probe 145 depicted in FIG.
6D, a cell-containing sample is introduced through sample
introduction element 163 and flows out onto matrix 145.
[0190] For all trapping enclosures 140 in which it is desired to
load cells 142, a functionally associated first conduit pump 190
and a second conduit pump 192 are set by central control device 144
to suction mode. A cell 142 in proximity of a respective open
trapping-end 170 is drawn into a respective trapping enclosure 140
and is drawn into a trapping enclosure 140 until making contact
with a respective cell-stop 184.
[0191] After a certain period of time when it is estimated that
cells are trapped in all desired trapping enclosures 140,
associated first conduit pumps 190 and second conduit pumps 192 are
set to inactive mode by central control device 144. Observation
component 150 receives a command from central control element 144
to examine all trapping enclosures 140 to ascertain if cells 142
are trapped therein. First conduit pump 190 and second conduit pump
192 of trapping enclosures 140 that are empty are again set to
suction mode until all desired trapping enclosures 140 hold cells
142.
[0192] Once cells 142 are held in all desired trapping enclosures
140, a waste pump functionally associated with a waste element is
set to suction mode by central control unit 144. Excess cells and
liquids not trapped in trapping enclosures 140 are removed and
disposed of through inlet 148 of the waste element. Subsequently,
fluid inlet pump 160 is activated to dispense an appropriate cell
culture medium through fluid inlet 154 onto matrix 145 by central
control unit 144.
[0193] In one embodiment, it is desired that cells 142 be trapped
in substantially all trapping enclosures 140. In such an
embodiment, first conduit pumps 190 and second conduit pumps 192
associated with all trapping enclosures 140 are set to suction mode
and consequently draw cells 142 into a respective trapping
enclosure 140.
[0194] In a different embodiment, it is desired to maintain a
maximal flexibility for movement of cells 142. In such an
embodiment, in a first step first conduit pumps 190 and second
conduit pumps 192 associated with only non-neighboring trapping
enclosures 140 are set to suction mode. In subsequent steps cells
can be moved and transferred between trapping enclosures with great
ease as every cell 142 has a free path to every trapping enclosure
140 of matrix 145, vide infra.
Cell Observation
[0195] In one embodiment of the present invention, a cell 142 in a
trapping enclosure 140 is observed through a respective open
trapping-end 170. In another embodiment, the walls of a trapping
enclosure 140 are transparent and a cell 142 held therein is
observed therethrough.
[0196] In a preferred embodiment, when cell manipulation elements
are provided with a conduit or trapping enclosure deep enough to
allow deep cell entry, cells 142 not to be observed are drawn
inside the cell manipulation element so that only one or only a few
cells 142, each in a trapping enclosure 140, remain in the
proximity of a respective open trapping-end. Observation is then
performed only on those cells 142 in proximity of a respective open
trapping-end 170.
[0197] Most preferably, if a cell 142 is held in a trapping
enclosure 140 of a cell manipulation element suitable for
suspending cell 142 above the trapping enclosure using the Venturi
effect (such as cell manipulation element 182), as described above,
then cell 142 to be observed is suspended for observation. Central
control unit 144 sets a conduit pump functionally associated with
axial conduit 186 to injection mode while conduit pumps
functionally associated with one or more peripheral conduits 174
are set to suction mode. As discussed hereinabove, cell 142 trapped
in trapping enclosure 140 is elevated out of trapping enclosure 140
and suspended thereabove. The advantages of suspending a cell 142
to be observed in such a way are manifold. Cell 142 can be
selectively and intensely illuminated using incoherent and/or
coherent light aimed at cell 142. Light scattering and shadows are
eliminated. Importantly the greater intensity of light allows the
use of a small camera aperture, increasing depth of field and
leading to the recording of a sharper image. Further, the intensity
of lighting can be chosen to maximize contrast and thus maximize
the observable details.
[0198] After cell 142 is examined, associated first conduit pump
190 and second conduit pump 192 may be both set to suction mode to
return cell 142 to trapping enclosure 140. Thereafter an additional
cell can be examined if required.
Reagent Addition through Vial Reagent Inlet
[0199] One way to expose cells to reagents is through a vial
reagent inlet 156. Central control unit 144 activates vial reagent
inlet pump 162 to dispense an amount of a reagent through a vial
reagent inlet 156 onto matrix 145. However, in order for a cell 142
held in a trapping enclosure 140 to come in contact with a
thus-dispensed reagent, the reagent must be brought into trapping
enclosure 140.
[0200] If all cells in all trapping enclosures are to be exposed to
the reagent (for example, in the case where the reagent is a
nutrient or a fluorescent marker), then one or both conduit pumps
190 and 192 associated with all trapping enclosures 140 are set to
suction mode, drawing an amount of reagent into each trapping
enclosure 140.
[0201] If, however, it is desired that only certain cells 142 be
exposed to a given reagent, then one or both conduit pumps 190 and
192 functionally associated only with the respective trapping
enclosures 140 of certain cells 142 are set to suction mode.
Reagent Addition through a Cell-Manipulation Component
[0202] As stated above, the fact that a given trapping enclosure
140 is functionally associated with two independently controllable
conduit pumps 190 and 192 allows "washing" of trapping enclosure
140 or of a cell 142 held within trapping enclosure 140 by using
one functionally associated conduit pump to inject a washing liquid
simultaneously with extraction of the washing liquid using the
other conduit pump.
[0203] As stated above, the fact that a trapping enclosure 140 is
functionally associated with two independently controlled conduit
pumps 190 and 192 together with a reagent injection pump 194 as
depicted in FIG. 7F allows selective exposure of a cell 142 in
trapping enclosure 140 to a reagent. Reagent injection pump 194 is
set to add a desired amount of reagent to the flow produced by
first conduit pump 190 is set to injection mode. The flow of fluid
from conduit 174 carries reagent to trapping enclosure 140, thus
exposing cell 142 to the reagent. To prevent leakage of the reagent
out of trapping enclosure 140, second conduit pump 192 is set to
suction mode, actively removing reagent and other fluids from
enclosure 140.
Moving a Cell to a Different Trapping Enclosure
[0204] The movement of a cell 142 held in a trapping enclosure 140
to another trapping enclosure by a series of transfers between
neighboring trapping enclosures is performed according to the
method of the present invention and is clear from the description
hereinabove. Such a transfer is schematically depicted in FIG.
11.
[0205] A cell 142 is held in a trapping enclosure of cell
manipulation element 236, FIG. 11A.
[0206] As depicted in FIG. 11A, conduit pumps functionally
associated with cell manipulation element 236 are set to injection
mode and conduit pumps functionally associated with neighboring
cell manipulation element 238 are set to suction mode. A flow of
liquid is produced from cell manipulation element 236 to
neighboring cell manipulation element 238, depicted as arrows
240.
[0207] As depicted in FIG. 11B, the force produced by flow of
liquid 240 ejects cell 142 from cell manipulation element 236 and
carries cell 142 towards neighboring cell manipulation element
238.
[0208] As depicted in FIG. 11C, cell 142 is ultimately carried into
the trapping enclosure of neighboring cell manipulation element
238.
[0209] A cell is moved from any trapping enclosure to any other
unoccupied trapping enclosure along a path defined by a series of
unoccupied neighboring enclosure, according to the method of the
present invention and as described herein and in FIG. 3. The
ability to move an individual cell from one trapping enclosure to
any other trapping enclosure allows the performance of many useful
experiments.
[0210] For example, it may be desirous to expose similar cells to a
series of reagents, but to compare the effect of changing the order
of exposure or the length of time a cell is exposed to a given
reagent. To perform such an experiment, cells are loaded into
trapping enclosures. The cells are sequentially transported to
different trapping enclosures provided with specific reagents and
therein exposed to reagents, as required.
[0211] It is also often desired to study the interaction of two
different types of cells. Cells of a first type are loaded into
trapping enclosures, preferably unoccupied trapping enclosures.
Cells of a second type are loaded into other trapping enclosures,
preferably unoccupied trapping enclosures. A cell of each type is
moved, as described above, to a multi-cell reaction enclosure 151.
In another embodiment contingent on a properly sized trapping
enclosure, a first cell is moved into a trapping enclosure already
occupied by a second cell. Observation component 150 is used to
observe any interaction. When desired, the two cells are ejected
from multi-cell reaction enclosure 151 and another pair of cells is
loaded therein for study.
[0212] It is also often desired to move a specific cell or cells
held in a trapping enclosure to an isolation element in order to
collect cells of a certain type for further study. Each individual
cell is transferred through a series of trapping enclosures, from
one trapping enclosure to a neighboring trapping enclosure, until
cell is located in a trapping enclosure 140 neighboring an
isolation inlet 146 of an isolation element. The cell is ejected
from trapping enclosure 140 while a conduit pump functionally
associated with the isolation element is activated so as to draw
cell 142 into the isolation element through isolation inlet
146.
[0213] Analogously, it may also be desired to discard a specific
cell 142 from a trapping enclosure. Cell 142 is transferred from
one trapping enclosure 140 to a neighboring trapping enclosure 140
until cell 142 is located in the vicinity of waste inlet 148. Cell
142 is ejected from trapping enclosure 140 while a pump
functionally associated with the waste element is activated so as
to draw cell 142 through waste inlet 148.
[0214] In most embodiments of the present invention, a device 130
is configured to allow simultaneous activation of many pumps and
thus perform manipulations and functions simultaneously. Thus, in
one embodiment of the present invention, numerous cells are
individually manipulated simultaneously using one device of the
present invention such as 130. Thus, in another embodiment of the
present invention, numerous cell-cell interactions are
simultaneously performed using one device of the present invention
such as 130. In another embodiment, numerous specific treatments
and manipulations of numerous individual cells are simultaneously
performed using one device of the present invention such as 130. In
another embodiment, many cells are simultaneously moved from a
first trapping enclosure to another trapping enclosure, as desired.
The preparation of an algorithm that calculates an optimal series
of steps, where in each step one or more cells 142 are transferred
from one trapping enclosure 140 to another, is well within the
ability of one skilled in the art of computer programming. Such an
algorithm is easily implemented in central control unit 144 of a
device 130 of the present invention. Once an optimal series of
steps is found, central control unit 144 sets the various conduit
pumps to perform the appropriate actions according to the
calculated series of steps so as to relocate and rearrange cells
142 as required.
[0215] Preferably, at any given moment, the locations of all cells
142 in trapping enclosures 140 of a cell manipulation probe 132 are
recorded in central control unit 144. Preferably, central control
unit 144 also keeps a record of the manipulations that any given
cell has undergone. In such a way, data can be easily analyzed.
Exemplary Experiments
[0216] One skilled in the art, upon perusal of the description of
the present invention hereinabove is able to construct a device of
the present invention and to plan a plethora of
heretofore-impossible experiments, manipulations and studies that
yield hitherto unavailable scientific information. For simplicity,
the words experiment, manipulation and study will be used
hereinbelow interchangeably. The manipulation of individual cells
enabled by the teachings of the present invention provides far more
information for the understanding of biological implications of
phenomena under study compared to bulk studies (e.g., using
cuvettes or microplates), flow-through cytometry studies or static
cytometry studies.
[0217] According to a preferred embodiment of the present
invention, specific cells are selected for real-time
high-throughput study. It is important to note that some of the
studies performed using the teachings of the present invention
including some of those described hereinbelow may have been
previously manually performed. It is important to note, however,
that the teachings of the present invention are the first that
allow the performance of such studies automatically on many cells
simultaneously as opposed to manually by highly skilled
individuals.
[0218] An additional advantage of the present invention is that
selected cells can be maintained for a relatively long period of
time in various environments, allowing a single cell to be used for
many different experiments. For instance, using a device of the
present invention a large number of cells is exposed to a stimulus
and cells that respond to the stimulus in a certain way are
isolated, for example, in trapping enclosures of a cell
manipulation probe or by transfer to an isolation element as
described above. Thereafter, the reaction of the isolated cells to
other stimuli can be studied. In summary, once a cell having
unusual or otherwise interesting properties is identified the
teachings of the present invention are used to isolate and further
manipulate the cell, thereby pinpointing an investigation to a
subgroup of relevant cells, reducing investigation time and saving
valuable resources. Additionally, since such an investigation is
done on a cell-by-cell basis, better and more precise data is
obtainable, for example, for increased diagnostic accuracy.
[0219] The teachings of the present invention can also be used for
sorting cells or selecting specific cells from a large population
of cells. Such procedures include the selective removal of
undesired cells or the harvesting of desired cells found in small
proportions from amongst other cells, activities that cannot be
performed using methods known in the art. Single-cell therapy can
be performed by identifying and isolating only cells exhibiting a
pathology from amongst a group of many cells, treating the isolated
cell in some fashion (e.g., exposure to a reagent) and returning
the thus-treated cell to the original group. Groups of cells having
exceptional interest include, for example, lymphocytes in blood, T
jurkat cell lines, lymph node cells, tumor cells and other
representative groups.
[0220] When applied to viable cells taken from a living organism
(such as a mammal, both human and non-human), the teachings of the
present invention enable the treatment of biological fluids from
living organisms (such as a mammal, both human and non-human) by
procedures including but not limited to the manipulation of
individual cells or removal of abnormal cells (especially harmful
abnormal cells) from amongst normal cells. Such a treatment can be
considered a form of selective manipulation of particles
(especially cells) in a biological fluid, including but not limited
to lymphatic fluids, blood, cerebral spinal fluids, semen, saliva,
synovial fluid, bone marrow, cochlear fluid, fluid extracted from
tumors, especially malignant tumors, ovarian fluid, amniotic fluid
and chorionic fluid. In one embodiment, such treatments can be
performed "off-line", that is by removing a sample of a biological
fluid from an organism (both human and non-human) treating (e.g.
selecting or manipulating cells) the fluid in a device of the
present invention and returning the treated fluid to the organism.
In a preferred embodiment, such treatments are performed "on-line",
that is by directly attaching an input line from the organism to a
device of the present invention, and returning the treated fluid to
the organism. An "on-line" treatment according to the present
invention of an organism is depicted in FIG. 12, where an input
line 243 is attached from an organism 245 to a device of the
present invention 247. In device 247 a desired treatment is
performed and the treated fluid is returned from device 247 through
an output line 249 back to organism 245. In such an embodiment it
is advantageous that the central control unit of device 247 be
configured to automatically detect and treat cells, for example by
using automated image analysis.
[0221] Another experiment that is performed using the teachings of
the present invention involves loading cells to be studied into
trapping enclosures of cell-manipulation elements equipped with
hollow penetrating-tools. The cell walls are penetrated as
described hereinabove and the penetrated cell inoculated with a
first reagent injected through the conduit in the
hollow-penetrating tool. The thus-prepared cell is exposed to a
second reagent in a manner as discussed hereinabove. The reaction
of the inoculated cell is then observed.
[0222] Another related experiment involves the improved
fertilization of ova (an improved form of IVF). A fluid sample
containing viable ova is loaded onto a cell manipulation probe of
the present invention. The ova are stored in trapping enclosures
equipped with hollow penetrating tools. The walls of the ova are
penetrated and sperm is injected therethrough into the ova. Each
individual ovum is released from the penetrating tool but retained
inside a trapping enclosure. Cell division and development is
observed and only ova that are successfully fertilized and
apparently viable are moved to an isolation element for transfer to
a uterus.
[0223] A device of the present invention can also be used to
transfer cells to a different device, for example a biochip
processor as described in PCT patent application PCT2001/IL 0000992
published as WO 03/035824. Cells are loaded and undergo
manipulation as described above including selection, sorting and
exposure to reagents and other stimuli. When it is so desired,
cell-manipulation probe 132 is brought in contact, in proximity or
somehow physically connected with a fluid passage (not depicted)
with the surface of biochip processor 242 (or the like such as a
microwell plate such as a Nunc 384-well plate) as depicted in FIG.
13A. A specific cell or cells are deposited from chosen trapping
enclosures 244 and 246 in cell-manipulation probe 132 to
identifiable locations 248 and 250 on biochip processor 242.
Biochip processor 242 is then used to handle the selected cells in
the usual way and as described in WO 03/035824. In such a way,
selected cells identified and isolated using the teachings of the
present invention may be grown and allowed to proliferate. In
another embodiment of the invention, this process is reversed so as
to load a cell-manipulation probe 132 of the present invention with
cells from a biochip processor 242 (or the like such as a microwell
plate such as a Nunc 384-well plate). Cell manipulation probe 132
is brought in proximity to the surface of biochip processor 242,
FIG. 13B. Associated conduit pumps are set to suction mode, loading
cells 142 from biochip processor 242 to trapping enclosures of cell
manipulation probe 132. The ability to easily couple two advanced
research methods exponentially increases the significant results
gleaned from an experiment. Such coupling is exceptionally
advantageous when the two research methods are format compatible,
that is, when the number, and preferably geometric arrangement, of
trapping enclosures in cell manipulation probe 132 are
substantially similar or even identical to the identifiable
locations (such as 248 and 250) of biochip processor 242.
[0224] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0225] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. For
example, it is important to note that for the sake of readability,
the term "pump" was used for the preferred device for the
production of flow in embodiments intended for use in fluids,
especially liquids. It is clear to one skilled in the art that the
intent herein is rather to a flow generator, that is, a device or
other means for generating a flow in a liquid.
[0226] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *