U.S. patent application number 14/154766 was filed with the patent office on 2014-05-08 for methods for improving in vitro measurements using boyden chambers.
This patent application is currently assigned to ESSEN INSTRUMENTS, INC.. The applicant listed for this patent is Essen Instruments, Inc.. Invention is credited to Bradley D. Neagle, Kirk Schroeder.
Application Number | 20140127744 14/154766 |
Document ID | / |
Family ID | 47293508 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127744 |
Kind Code |
A1 |
Schroeder; Kirk ; et
al. |
May 8, 2014 |
METHODS FOR IMPROVING IN VITRO MEASUREMENTS USING BOYDEN
CHAMBERS
Abstract
Apparatus and methods to improve the Boyden chamber used in
cellular biological measurements, allowing quantitative optical
microscopy of biological cells in situ without using fluorescent
probes or optical staining. In the preferred embodiment, a thin
porous membrane separating top and bottom reservoirs includes an
array of precisely positioned micropores pores manufactured using a
laser-based photo-machining (ablation) process. The membrane may be
composed of polyethylene terephthalate (PET), polycarbonate,
polyimide, polyether ether ketone (PEEK) or other appropriate
material. The pores formed in the membrane may have diameters in
the range of 1 to 15 microns and spaced apart at a distance ranging
from 10 to 200 microns. A plurality of upper and lower reservoirs
may be provided to form a multi-well plate. The invention finds
application in a wide range of potential biological applications
where Boyden chamber geometries are currently used including
co-culture studies, tissue remodeling studies, cell polarity
determinations, endocrine signaling, cell transport, cell
permeability, cell invasion and chemotaxis assays.
Inventors: |
Schroeder; Kirk; (Ann Arbor,
MI) ; Neagle; Bradley D.; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Essen Instruments, Inc. |
Ann Arbor |
MI |
US |
|
|
Assignee: |
ESSEN INSTRUMENTS, INC.
Ann Arbor
MI
|
Family ID: |
47293508 |
Appl. No.: |
14/154766 |
Filed: |
January 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13157873 |
Jun 10, 2011 |
8673628 |
|
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14154766 |
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Current U.S.
Class: |
435/39 ;
435/29 |
Current CPC
Class: |
G01N 33/54366 20130101;
G01N 21/6458 20130101; G01N 33/5008 20130101; B01L 3/5085 20130101;
B01L 2200/0647 20130101; G01N 33/58 20130101; B01L 2300/0681
20130101; C12Q 1/02 20130101; G01N 21/6452 20130101; B01L 3/50255
20130101; B01L 2200/142 20130101; B01L 2300/0654 20130101; G01N
33/5029 20130101; B01L 2300/0829 20130101; B01L 2300/163
20130101 |
Class at
Publication: |
435/39 ;
435/29 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A biological measurement method, comprising the steps of:
providing a thin film membrane; forming a plurality of micropores
in the membrane using a laser-based photo-machining (ablation)
process; using the membrane to separate upper and lower
fluid-containing reservoirs; and performing quantitative optical
imaging of biological cells on the membrane without using
fluorescent probes or optical stains.
2. The method of claim 1, wherein the step of imaging is performed
with one of Zernike phase contrast, differential interference
contrast (DIC) or Hoffman modulation contrast.
3. The method of claim 1, further including the use of
epifluorescence microscopy.
4. The method of claim 1, wherein the method is used for the
measurement of cell migration (chemotaxis), cell invasion, cell
permeability, tissue remodeling, cell polarity endocrine signaling
or cell transport.
5. The method of claim 1, wherein the step of quantitative imaging
involves a morphological assessment of shape, and/or the counting
of the cells on the surface of the membrane and/or the
identification of a particular cell type within a mixed cell
population.
6. The method of claim 1, wherein the step of counting the cells
remaining on the top side of the membrane at multiple time points
is used to quantify the amount of cell chemotaxis or cell
invasion.
7. The method of claim 1, wherein kinetic, multi-time point
quantitative optical microscopic measurements are to reduce
artifacts associated with transient chemical gradients in
chemotaxis or chemo-invasion assays.
8. The method of claim 1, wherein the step of forming a plurality
of micropores in the membrane includes forming pores with diameters
in range of 1 to 15 microns and spaced apart at a distance ranging
from 10 to 200 microns.
9. The method of claim 1, wherein the step of providing a thin film
membrane includes providing a polyethylene terephthalate (PET),
polycarbonate, polyimide or polyether ether ketone (PEEK) thin
film.
10. The method of claim 1, including the step of fabricating a
plurality of porous membranes, each separating a respective upper
and lower reservoir, thereby forming a multi-well plate.
11. The method of claim 1, including the step of: coating the
membrane with collagen 1, fibronectin, laminin or other
extracellular matrix.
12. The method of claim 1, including the step of: forming upper and
lower reservoirs using injection-molded polystyrene, polycarbonate
or polyethylene.
13. The method of claim 1, including the step of: ultrasonically
welding or chemically bonding the porous membrane to the upper or
lower reservoir.
14. The method of claim 1, including the step of: attaching the
membrane to the bottom surface of the top reservoir, thereby
forming a removable insert that fits inside the bottom
reservoir.
15. The method of claim 1, including the step of: optimizing the
pore diameter, pore locations and timing of data acquisition in
order to reduce artifacts associated with a transient diffusion
gradient in chemotaxis or chemo-invasion assays.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/157,873, filed Jun. 10, 2011, the entire content of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to cellular biological
measurement including cell migration (chemotaxis), cell invasion,
cell permeability, tissue remodeling, cell polarity endocrine
signaling and cell transport and, in particular, to apparatus and
methods for improving the Boyden chamber apparatus used in such
measurements.
BACKGROUND OF THE INVENTION
[0003] Cell migration is critical in many physiological processes.
Chemotaxis, for example, is the study of cell motion in response to
a soluble chemo-attractant stimulus. Similar and related mechanisms
include haptotaxis and chemoinvasion, which rely on cell motility
on a substrate-bound stimulus and movement through an Extracellular
Matrix (ECM) boundary layer, respectively. These processes play a
vital role in the study of many therapeutic areas including
oncology, inflammation and angiogenesis. In recent years there have
been many advances in the understanding of this physiological
response, and much work has been done on the various classes of
cytokines (e.g. TNFa, IL-1) and chemokine receptors (e.g. CCR2,
CCR5).
[0004] The most common technique for measuring cell migration in
vitro is via a measurement geometry known as the Boyden chamber [1]
first described in 1962. This geometry, developed by Dr. Stephen
Boyden consists of two chambers separated by a porous membrane as
depicted in FIG. 1. When using a Boyden chamber as a chemotaxis
measuring device, a chemoattractant is typically added to the lower
chamber, cells are added to the upper chamber and the porous
membrane serves as a means to establish a diffusion-based chemical
gradient between the upper and lower chambers. The cells on the top
side of the membrane detect this gradient, migrate to the
individual pores in the membrane, and then crawl through the holes
to the lower chamber. Once migrating through the pore, the cells
ultimately either fall through the membrane to a lower reservoir,
or end up migrating on to the bottom side of the membrane.
Determination of the chemotactic response relies on quantifying the
number of cells that migrated through the membrane in relation to
the total number of cells added to the top chamber.
[0005] There have been many small improvements to the standard
Boyden chamber geometry since its inception, but the basic device
geometry and membrane components have remained fairly consistent.
Currently, there are many commercial sources for 6-well, 24-well or
96-well Boyden chamber-derived chemotaxis kits, including the
ChemoTx.TM. system sold by Neuroprobe Inc. (Gaithersburg, Md.), the
Transwell.TM. system sold by Corning Life Science (Acton, Mass.)
and the HTS Fluoroblok.TM. system sold by Becton-Dickinson
(Franklin Lakes, N.J.). All of these devices are basically
rectangular arrays of Boyden chambers using microplate formats and
injection mold fabrication techniques for the upper and lower
reservoirs. All of these commercial devices also use the same basic
material for the porous membrane which is known as a "Track-Etch
Membrane".
[0006] Track-etch membranes are manufactured by exposing thin
polymer films (e.g. polyester, or polycarbonate) to radioactive
particle bombardment, followed by chemical etching [2]. The results
of this manufacturing process are a thin film with a random pattern
of very defined micro-holes as shown in the brightfield image of
FIG. 2. The density of micro-holes using this fabrication technique
is governed by the exposure time and exposure geometry in relation
to the radioactive source. The size of the micro-holes in a given
track-etch membrane is controlled by a combination of time,
temperature and the chemical concentration used during the etch
step. Typical etch solutions include highly concentrated NaOH, or
HF. The micro-hole size is very uniform, and in general fairly
orthogonal to the surface of the membrane. Pore size typically
ranges from 0.2 microns, up to 10 microns in diameter. For
filtering applications, it is generally better to have higher pore
densities. However, due to the random nature of the ionization
particle bombardment, pore density has a practical upper limit so
as to avoid the random occurrence of "doublets", i.e. two pores
touching each other. As such the "porosity" of track-etched
membranes, defined as the area of pores divided by the area of
non-pore, is generally on the order of a few percent.
[0007] The predominant application of such membranes is for fine
particle and contaminant filtering of fluids as well a method of
capturing and detecting microorganisms. The filtering applications
take advantage of the very uniform and defined pore size of the
membrane. This characteristic makes these types of membranes ideal
for precisely filtering particles or micro-organisms of a given
size. For biological applications such as cell migration,
track-etch membranes of 2, 3, 5 and 8 micron diameter pore sizes
are most commonly used. For these applications, pore size is often
matched to size of the cells being studied, bigger cells use bigger
pores. Typically, the pore size is chosen to be slightly larger
than the nucleus of the cells being studied. Most of the commercial
manufacturers of Boyden-style chemotaxis chambers offer a variety
of products incorporating different pore sizes. They also offer
membranes in different materials, most commonly polycarbonate and
polyester, and supply biological substrate coatings (e.g. collagen,
fibronectin or laminin) or surface coating protocols. These
biological coatings are sometimes useful so as to more accurately
mimic the in vivo surface/adhesion biology of migrating cells.
[0008] The quantitative read-out from a Boyden chamber chemotaxis
assay is based on a count of the number of cells which migrate
through the membrane towards the chemoattractant in the lower
chamber. Quite often, one must also employ negative control
measurements where no chemoattractant is used in the lower chamber
to correct for random migration effects. In existing commercial
Boyden chamber technologies, quantification of the number of cells
is accomplished by using fluorescent dye labeling of the cells.
Labeling of the cells is necessary as the cells cannot be
visualized on the surface of the track-etch membranes directly
without using a fluorescent marker. Once labeled, the cells can be
counted directly using cell counting microscopy; or if a
proportionality relationship can be established between
fluorescence and cell number, a bulk fluorescent measurement can be
made which is proportional to cell number. Cells are typically
labeled at the beginning of the experiment, i.e. before cell
migration occurs. Cells can also be "post-labeled", i.e. after the
cell migration occurs. This latter method is often preferred when
working with time-sensitive or label-sensitive cell types.
[0009] Boyden chamber technology is the current "gold standard" for
in vitro chemotaxis assays and has been around for almost fifty
years. The modern incarnations of the technique have the advantage
of being amenable being amenable to multi-well microplate formats
and the precision of plastic injection molding techniques; as such
they are fairly high throughput and reasonably priced. While being
the current gold standard, and clearly dominating the research
market, there are several disadvantages to the current Boyden
chamber systems. These disadvantages will be discussed in the
following paragraphs
DISADVANTAGES OF PRIOR ART BOYDEN CHAMBER GEOMETRY FOR MEASURING
CHEMOTAXIS
A. Temporal Variation of the Chemoattrant Gradient:
[0010] Because Boyden chambers rely on passive chemical diffusion
in order for a gradient to be formed between the upper and lower
reservoirs, there is no active control of the gradient process. The
chemical gradient formed is simply a function of the pore size,
time, concentration and the molecular weight of the
chemoattractant. The gradient is, by definition, time varying and
eventually the top chamber and bottom chamber chemical
concentrations equalize and the gradient is destroyed. Not having
the ability to modulate or control the time varying aspect is a
disadvantage of this approach. In addition, because of the random
spatial location of the pores formed by the track-etch process, the
chemical diffusion gradient is also very non-homogeneous at the top
surface of the membrane. Lastly, the transient and non-homogeneous
nature of the chemical gradient using current methods can cause
time-sensitive, and concentration cell migration responses. These
in turn can cause artifacts where, depending on the timing and or
concentration of the agonist, it is impossible to determine if a
reduction in cell migration is due to a degradation of the chemical
gradient, or the effect of an experimental pharmacological
agent.
[0011] In recent years, researchers have designed and built
chemotaxis chambers using microfluidic channels with the goal of
establishing better defined chemical gradients as well as adding
the ability to image the migrating cells directly. A few of these
approaches have reached the commercial market [3,4]. These
microfluidic approaches generally allow for a more quantified
chemical gradient to be formed, offering the ability to
characterize the formation and temporal characteristics of the
gradient using fluorescent labeled molecules and fluorescent
microscope detection. However, although the gradients are
quantitatively characterized, none of the micro-fluidic approaches
have eliminated the temporal and transient nature of the gradient
formation and the potential artifacts this can cause.
[0012] Also, micro-fluidic devices work with small fluid volumes.
As such getting fluids and living cells into micro-fluidic chambers
is cumbersome, and typically researchers end up working with very
small fluid volumes which are prone to evaporation effects.
Physiological buffering agents can cause osmotic stress to living
cells in the presence of even minor amounts of evaporation. The
smaller the volumes utilized the more sensitive the effects of the
evaporation can become. Another drawback of the micro-fluidic
approaches is that they tend to provide very shallow chemical
gradients, on the order of a percent or two of absolute
concentration change across a cell diameter. It is generally
believed that a cell requires at least a 2% absolute concentration
difference across the cell diameter in order for the cell to
respond in a directed manner. The concentration gradients achieved
in microfluidic devices are on this order or smaller, as such the
microfluidic solutions tend to be limited in their chemotactic
efficiency (the number of cells which respond). So while there are
some advantages to the microfluidic approaches, to date, none of
the microfluidic-based commercial solutions has supplanted the
Boyden chamber has the dominant "gold standard" measurement system
for in vitro chemotaxis.
B. Current Techniques Require Fluorescent or Optical Dye Labeling
to the Cells:
[0013] Quantitation of chemotaxis in a Boyden chamber geometry
relies on fluorescently labeling the cells, either prior to the
experiment, or after the experiment in a post-labeling step. The
"signal" from a Boyden chamber chemotaxis assay relies on counting
the number of cells that have migrated from the top side of the
membrane through the micro-hole of the track-etch membrane. Once
migrating through the pore, some cell types will adhere and migrate
to the bottom side of the membrane. Other cell types will not
adhere, and will fall through to a bottom collection chamber,
typically a reservoir microplate. Cells are "counted" either by
using a fluorescent microscope and "counting" individual cells on
the bottom side of the membrane, or those in the bottom reservoir
(or both). In some cases, a microscope is not used but rather a
bulk fluorescent measurement is made using a fluorescent plate
reader. This latter technique relies on establishing a fluorescent
calibration curve between the fluorescent label used and the number
of cells.
[0014] The practical impact of requiring a fluorescent label to
effectively "count" the cells is a big disadvantage in running
these types of assays. First and foremost, a lot of chemotaxis
assays rely on using "primary" hematopoetic blood cells (e.g.
T-lymphocytes, neutrophils) which are very sensitive to potential
toxic effects of fluorescent dyes. Secondly using fluorescent or
optical dye labeling techniques makes the assays more expensive as
the labeling step and reagents cost time and money. Lastly, the
presence of labels can make it is necessary to introduce
non-homogeneous work-flow processing steps. For example, if the
cells migrate to the bottom of the track-etch membrane, it may be
necessary to manually remove or `scrape" any remaining fluorescent
cells from the top side of the membrane to avoid counting these
cells as migrated cells. Any manual process like cell scraping
takes more time, interrupts the workflow, thereby limiting assay
throughput, and introducing dominant errors in the quantitation of
the response.
[0015] Biological cells are in general transparent, and rely on
either fluorescent labeling to view or host of other techniques
which encode subtle optical phase changes introduced by the cells
into detectable intensity change. Imaging techniques which work on
this premise include Zernike phase contrast, differential
interference contrast (DIC) and Hoffman Modulation Contrast
(Phase-Sensitive Techniques). Phase-sensitive imaging techniques
rely on placing the cells on very high optical quality substrates
such as thin glass slides or thin plastic substrates. It is not
enough however, that the substrate be optically clear, it must also
not introduce any significant phase perturbation to the optical
wavefront which would distort or mask the subtle phase variations
introduced by the biological cells. Phase-sensitive imaging is not
amenable to the current commercially-available Boyden chamber
consumables which rely on porous membranes manufactured by the
track-etch manufacturing process. Membranes produced by the
track-etch process introduce a variety of optical phase
perturbations when imaged with a phase contrast microscope, making
detection of the cells on the surface without using fluorescent
labels or optical dyes/stains impossible as shown in FIG. 3. As
such, commercially available Boyden chamber geometries are not
amenable to direct cell viewing.
[0016] Cells undergoing migration take on many distinctive
morphological phenotypes often denoted as "shape change". The
ability to detect "shape change" as well as to validate the
chemotactic signal that one is measuring during a chemotaxis assay
without fluorescently or optically labeling the cells would be
extremely beneficial. In fact, the ability to visualize the cells
during the assay has been one of the key design motivations for the
development of the micro-fluidic approaches aforementioned.
Unfortunately this advantage has not overcome the disadvantages
associated with relatively shallow chemical gradients and
evaporation effects.
C. Number of Cells Required to Quantify the Response:
[0017] Another disadvantage of currently available Boyden chamber
derived chemotaxis kits is that they require large number of cells
to characterize the response, typically tens to even hundreds of
thousands of cells per well. Large cell numbers can be a major
cost/adoption disadvantage. This is especially the case when using
rare, and perhaps difficult to isolate primary hematopoietic cells
from the blood.
[0018] One reason that commercially available Boyden chamber
technologies require large cell numbers per well, is that at any
given concentration gradient, the actual "participation rate", i.e.
the number of cells which may respond to the level of chemical
gradient may be fairly small (e.g. a few %) of the starting number.
In order for the experiment to result in a reasonable number of
cells migrating through the porous membrane, one has to start with
very large numbers of cells on the top side of the membrane. The
dynamic range problem is further complicated in that often
researchers are interested in finding compounds which inhibit the
chemotactic response. The result is that a low signal level gets
even smaller upon inhibition, thereby creating a small dynamic
range ("signal window") for the assay.
[0019] Detection of very small cell numbers of migrating cells from
the starting population is difficult to do with current commercial
systems as it is not easy to image the various surfaces where the
cells are located (top side of membrane, bottom side of membrane or
lower reservoir). If using fluorescence, one must be able to
separate fluorescent signal from the top side of the membrane from
fluorescent cell counts from the bottom side of the membrane. This
may require the need for time-consuming, manual assay steps such as
"manual cell scraping", in order to separate the fluorescent signal
on the top side of the membrane vs. the fluorescent signal on the
bottom side of the membrane. These types of steps are prone to
human error and variability and are greatly detrimental to the
precision of a Boyden chamber chemotaxis assay.
[0020] To eliminate this problem, Becton Dickinson (BD) introduced
the Fluoroblok.TM. technology in 1997 (U.S. Pat. No. 5,601,997).
This invention introduced an optically-opaque barrier in the
membrane, such that detection of the fluorescence from the bottom
side of the membrane would not be sensitive to any
fluorescently-labeled cells remaining on the top side of the
membrane. This was a definitive advance in helping to remove manual
preparation steps. Unfortunately, the Fluoroblok.TM. consumables
are relatively expensive, the technique still requires fluorescent
labels, it is not easy to image the cells or observe cell
morphology and the systems require large numbers of cells per well.
Assays using the BD Fluoroblok.TM. technology typically require
50,000 to 100,000 cells per well [5].
[0021] In summary, the Boyden chamber geometry remains the
gold-standard measurements technique for measuring in-vitro
chemotaxis. However, the commercial solutions for the Boyden
chamber geometry all suffer from the following disadvantages:
[0022] 1.) Time varying chemical gradient
[0023] 2.) No ability to alter spatial pore geometry
[0024] 3.) Require fluorescent probes or optical dyes of the cells
to quantify the response
[0025] 4.) Not amenable to viewing the cells in situ during the
assay
[0026] 5.) Require large numbers of cells
[0027] Microfluidic chemotaxis assay designs have improved on the
ability to directly image the cells, and have incorporated the
ability to characterize the chemical gradient formation, however,
they suffer from:
[0028] 1.) Resulting chemical gradient is very shallow
[0029] 2.) Detrimental evaporation effects, osmotic stress
effects
[0030] 3.) Are either extremely low throughput, or cumbersome to
use
SUMMARY OF THE INVENTION
[0031] This invention relates to an apparatus and methods for
improving the Boyden chamber used in cellular biological
measurements. In accordance with the invention, cells can be
directly imaged and analyzed in situ and without using fluorescent
labels or optical dye staining. In addition, the pore spacing and
pore diameter of the filter membrane can be precisely controlled,
enhancing the use of the devices for chemotaxis and chemo-invasion
type assays.
[0032] Apparatus constructed in accordance with the invention
includes a bottom reservoir, a top reservoir, and a thin porous
membrane separating the top and bottom reservoirs. In the preferred
embodiment, the pores of the membrane are manufactured using a
laser-based photo-machining (ablation) process.
[0033] The membrane may be composed of polyethylene terephthalate
(PET), polycarbonate, polyimide, polyether ether ketone (PEEK) or
other appropriate material with optical characteristics which do
not introduce significant phase perturbation to an incoming light
wavefront in relation to those introduced by biological cells on
the surface of the membrane. The pores formed in the membrane may
have diameters in the range of 1 to 15 microns and spaced apart at
a distance ranging from 10 to 200 microns. A plurality of upper and
lower reservoirs may be provided to form a multi-well plate.
[0034] The invention may be used for the measurement of cell
migration (chemotaxis), cell invasion, cell permeability, tissue
remodeling, cell polarity endocrine signaling or cell transport. In
a common application the porous membrane is used to separate upper
and lower fluid-containing reservoirs and coated with collagen 1,
fibronectin, laminin or other extracellular matrix material. An
inverted phase contrast, DIC, or Hoffman Modulation-type microscope
may be used to assess the morphology and/or number of biological
cells on the membrane. An advantage of the invention is that
chemotaxis, cell migration, cell invasion and other processes may
be carried out by counting the cells on the top surface of the
porous membrane directly and without using fluorescent labels or
optical dye staining.
[0035] The improvements made possible by the invention impact a
great range of potential biological applications where Boyden
chamber geometries are currently used including co-culture studies,
tissue remodeling studies, cell polarity determinations, endocrine
signaling, cell transport, cell permeability, invasion and
chemotaxis assays. While the description presented here is intended
to describe the advantages of the invention specifically as it
relates to the measurement of in vitro chemotaxis and in vitro cell
invasion assays, many of the attributes associated with the
invention are extendable to other common uses of Boyden chamber
systems.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 depicts a single well of a prior-art Boyden
chemotaxis chamber geometry;
[0037] FIG. 2 shows a prior-art bright field image of a track-etch
membrane surface;
[0038] FIG. 3 illustrates a preferred embodiment of a Boyden
chamber using a photo-machined membrane;
[0039] FIG. 4 shows a rectangular array of micro-holes forming the
porous membrane of a Boyden chamber geometry;
[0040] FIG. 5 is an inverted phase contrast imaging geometry for
the improved Boyden chamber;
[0041] FIG. 6A illustrates a phase contrast image of a track-etch
membrane (no cells);
[0042] FIG. 6B illustrates a phase contrast image of a track-etch
membrane (with cells);
[0043] FIG. 6C illustrates a phase contrast image of cells on a
high quality plastic substrate;
[0044] FIGS. 7A and 7B are graphs illustrating an agonist
saturation effect for existing Boyden chamber chemotaxis
assays;
[0045] FIGS. 8A and 8B depict a side view and a top view of a
preferred embodiment of the invention including a 96-well
consumable comprised of 96 individual Boyden chambers using a laser
photo-machined membrane; and
[0046] FIGS. 9A-9D show potential well geometries for reducing cell
usage.
DETAILED DESCRIPTION OF THE INVENTION
[0047] This invention is broadly directed to a process of
manufacturing the porous membrane used in Boyden chamber design.
Traditional track-etch membranes used in the production of
commercially available Boyden chamber devices commonly used thin
polymer films such as polycarbonate or polyethylene terephthalate
(PET). The process of making pores in track-etch membranes involves
bombarding the surface with radiation, typically alpha particles
followed by a chemical etching step using highly concentrated NaOH
or HF acid. The pore density (number of pores and density) is
controlled by the physical geometry and exposure levels to the
radiation source. The pore size is determined by the concentration
and exposure time in the etch step. Unfortunately, the result of
this process is a material that has significant optical surface
blemishes. These blemishes prohibit the use of common non-labeled,
non-invasive imaging techniques such as phase contrast, or
differential interference contrast (DIC) to view cells on the
surface of the membranes.
[0048] FIG. 1 shows a Boyden Chamber single-well geometry for
measuring cell migration utilizing a track-etch membrane filter.
Migrating cells can either adhere to the lower side of the
track-etch membrane, or fall through the membrane to a lower
reservoir for detection. FIG. 2 is a bright-field image of a
track-etch membrane. Micropores are random. One of the enabling
aspects of the disclosed invention is the ability to precisely
locate the individual micro-holes. Surface and material
characteristics make non-labeled, phase contrast imaging of cells
on the surface impossible. All that is visible without using
fluorescent labels are the micro pores themselves.
[0049] The preferred embodiment of this invention resides in a
porous membrane manufactured by a different manufacturing process,
one based on laser-based photo-machining. Laser-based
photo-machining generally uses a pulsed laser of the proper
wavelength and pulse energy such that the optical energy is
absorbed by the target material (in this case a thin polymer film)
resulting is small pieces being ablated from the surface with each
pulse. The laser beam can be precisely positioned in X-Y and the
beam dimension, optical power and pulse repetition frequency can be
precisely controlled so as to allow for very fine micro-hole
machining. The goal is to provide a regular-spaced grid of
micro-holes in a thin film whereby the pore density and pore size
are carefully controlled and the resulting porous material has much
improved optical surface characteristics in comparison to
track-etch membranes (prior art).
[0050] FIG. 3 depicts one specific embodiment of the invention,
where the track-etch membrane is replaced by photo-machined
membrane. It is envisaged that much like current track-etch
membranes, a variety of micro-hole sizes and densities could be
utilized to optimize a measurement geometry for a particular cell
type and assay. One of the advantages of this approach, is being
able to control the precise location and density of the
photo-machined micro-holes. This is in contrast to the random
location of the microholes as provided with track-etch membranes
(prior art).
[0051] For chemotaxis studies, it is often desirable to scale the
size of the micro-holes in the membrane in proportion to that of
the cells being analyzed. This scaling helps to alleviate the
contribution of chemokinesis (random migration) to the measured
chemotactic signal. Using photo-machining to make the individual
micro-holes provides, for the first time, the ability to control
the micro-hole density and spatial location. FIG. 4 is a specific
example of a preferred embodiment using a 16.times.16 array of
microholes, 8 uM in diameter and 60 microns apart. The specific
dimensions shown here are only an example, demonstrating the fact
that unlike the track-etch membranes, the pore density can be
carefully controlled. Using this approach, one could design and
implement many defined variations of micro-hole size and spatial
density.
[0052] The ability to laser photo-machining (ablation) of the
micro-holes to fabricate the porous membrane has many potential
advantages. First and foremost, the photo-machining process is not
detrimental to the optical quality of the thin film polymer
material. Unlike the track-etch membrane process which requires a
chemical etch of the entire surface to form the pores, the laser
machining process has minimal detrimental impact on the optical
quality of the film in the regions between the micro-holes. The
laser machining process is local machining process, whereby some
ablated material will be deposited around the hole, but this has
very little detrimental effect on the optical quality of the
polymer film in the area between the micro-holes. Maintaining a
high optical quality substrate enables quantitative, high contrast
morphological analysis and/or cell counting of individual living
cells on the membrane without using fluorescent labels or optical
dyes. This is a big advantage over current Boyden chamber
methodology which requires fluorescent labeling to image and count
the cells due to the poor optical characteristics of the track-etch
membranes. FIG. 6A is a phase-contrast image of a prior art
track-etch membrane. FIG. 6B is a phase contrast image of the
track-etch membrane with cells on the surface of the membrane. Due
to the optical perturbations caused by the membrane, quantitative
imaging is not possible. FIG. 6C is a phase contrast image of
cancer cells on a high quality optical substrate, tissue culture
plastic in this example. FIG. 5 is an example of inverted of an
inverted phase contrast method applied to a single well of a Boyden
chamber geometry using a photo-machined membrane. Precise
photo-machining of the thin polymer film leaves an optical surface
free from aberrations and enables the use of phase contrast, or
other non-labeled, imaging techniques for enumerating the cells on
the membrane in situ. This imaging geometry cannot be used to
quantitatively assess cell biology with current commercially
available Boyden chamber solutions using track-etched membranes as
the cells are not visible as demonstrated in FIG. 6C.
[0053] The ability to image the cells on the membrane directly has
many potential benefits aside from being able to quantify cell
migration without the use of labels. A non-labeled, homogeneous
assay approach, is amenable to a kinetic, multiple time point
read-out. Often, labeling of the cells precludes kinetic read-outs
due to the fact that the cells are dye loaded after the cell
migration is over, or if pre-loaded, the dyes can introduce
phototoxicity upon repeated light exposure. Using a non-labeled
approach eliminates manual intervention steps such as scraping
cells off the top, or bottom side of the membrane in order to count
them. These types of manual steps are prone to human error and
subjectivity, and cause errors in the precision of the assay.
Making the assay protocol homogeneous, allows for better assay
controls, and in-turn should reduce the number of cells required to
quantify the migration response.
[0054] Not having to label the cells, in combination with an
optimized micro-hole spacing, can also be used to reduce the
detrimental effects of the transient nature of the chemical
gradient formation. It is well known that Boyden chamber assays are
associated with bell-shaped pharmacological response to a
chemoattractant as is shown in FIGS. 7A and 7B which depict
"agonist" bell-shaped curves observed with current commercially
available Boyden chamber measurements. Shown is the number of cells
undergoing chemotaxis (vertical axis) versus an increasing
concentration of chemoattractant, IL-8 in FIG. 7A and C5a in FIG.
7B. Higher concentrations are non-optimum and can appear the same
as inhibition effects one is trying to measure. It is anticipated
that having a kinetic, homogenous assay read-out along with control
of the spatial hole geometry will provide an enhanced ability to
isolate this artifact. Data from [8] Frevert et al.
[0055] As chemoattractant "agonist" concentration is increased, one
measures and increase in chemotactic response up to a maximum
response. Increasing concentrations further causes the chemotactic
signal to actually decrease. It is theorized that this is the
result of an oversaturation of chemoattractant on the top side of
the Boyden chamber, effectively confusing the cells and causing a
muted response. This concentration and time-dependent effect makes
determination of antagonist pharmacology very difficult, as slight
deviations from the optimum "peak" of the agonist curve cannot be
easily distinguished from those of a competitive antagonist which
should also be inhibiting the response. It is our believe that
having control of the spatial micro-hole density, using a kinetic,
non-labeled read and homogeneous assay format will allow
researchers to better control this effect and thereby provide more
robust antagonist pharmacology.
[0056] Lastly, much like the design attribute of micro-fluidic
channels, having the ability to directly view the cells during the
chemotaxis process allows researchers to study morphological
changes, and or associate the response to other imaging parameters
for example the "shape change" associated with a migrating or
invasive morphological phenotype. Aside from the phase contrast or
DIC image, improved optical clarity will also improve, though not
enable, the use of fluorescence probes. One salient example would
be the combination of phase-sensitive imaging with fluorescent
labels for sorting out the effects of mutant cell lines, or
identifying different migration parameters for mixed cell lines.
However, unlike the use of micro-fluidic channels, the invention
presented here is easy to use, does not suffer from evaporation
effects or shall chemical gradients.
[0057] It should be understood, that the single-well geometry
described in FIGS. 3, 4 and 5 can easily be extended to a
rectangular array comprised of a plurality of wells and common
formats used the biological sciences for example 6-well, 24-well,
96-well, 384-well and 1536 well geometries. An example of a 96-well
format is depicted in FIG. 8A and FIG. 8B. Each of the individual
wells would have its only two-dimensional grid of laser machined
micro-pores. The commercial solutions previously described are
generally found in 6-well, 24-well and 96-well formats. One of the
design improvements in our invention is to be able to make a
quantitative measurement using far fewer cells. To achieve this we
could envision using a smaller reservoir dimension than those
currently available as demonstrated with the examples shown in
FIGS. 9A-9D. FIG. 9A shows individual well dimensions for a
standard 96-well format. The standard 96-well format uses 6 mm
diameter wells in a 12.times.8 format on 9 mm centers and is the
highest format commercially available. A preferred embodiment of
this invention would be to use smaller well dimensions so as to
reduce cell usage required in current commercially available Boyden
chamber systems. FIGS. 9B, 9C and 9D demonstrate different well
geometries with different micro-hole patterns and densities. The
individual wells shown could be placed on 9 mm centers to be
consistent with a 96-well geometry, or could be utilized in a 384
well or higher density microplate format.
[0058] Although the invention described in this document is
directed to a test chamber for measuring the migration of cells to
chemical stimuli, e.g., chemotaxis or chemokinesis, the invention
has applications beyond cell migration for example in the
measurement of cell permeability, cell transport, cell invasion
(others) where direct imaging of the surface would allow for
non-invasive, quantitative assessment of the cells on the membrane
in situ.
Alternative Embodiments
[0059] Different types of polymers and polymer thicknesses,
amenable to the laser ablation photo-machining may be used in
accordance with the invention. Variations in pore size, pore
density and pore location are also anticipated. It is anticipated
that pore geometry and spacing will be used to enhance the gradient
homogeneity at the top surface of the membrane. Various imaging
systems could be used to image the membrane in situ, including
epifluorescence, iZernike phase contrast, differential interference
contrast (DIC), Hoffman modulation contrast and others. Different
data processing schemes could also be utilized including measuring
just the cells on the top side of the membrane, or alternatively
measuring cells at three different planes being the a) top side of
the membrane, b) bottom side of the membrane and at the bottom of
the collection reservoir (lower chamber).
[0060] In order to reduce cell usage, many different potential
reservoir configurations are possible, including moving to smaller
reservoir formats, or smaller microplate formats such as a
half-area 96-well format, 384-well format, or 1536-well format.
Another alternative embodiment would be to apply biological
coatings to the surface of the membrane such as collagen 1,
fibronectin, or laminin. This type of coating would be a very thin
molecular surface coating so as not to plug the microholes. It is
also possible to apply a thicker extracellular matrix (ECM) coating
to the membrane, where the Boyden chamber can be used to measure
the ability of cells to invade the ECM, an assay known as a cell
invasion assay. This is a natural extension and common use of
existing Boyden chamber consumables.
[0061] There are several advantages of this invention over existing
prior-art Boyden chamber devices. While much of this document has
described the advantages for measurements of cell migration, many
of these advantages are extendable to other applications for Boyden
chambers. This, non-obvious modification, has several practical
benefits: [0062] a) Replacing the track-etch membrane of the Boyden
chamber with one manufactured by laser ablation, enables for the
first time in situ quantitative optical imaging methods such as
inverted Zernike phase contrast, differential interference contrast
(DIC) and Hoffman modulation contrast techniques to be used. [0063]
b) Cells no longer have to be exposed to potentially invasive,
fluorescent labeling dyes or protocols. This is very important for
primary cells which can be time-sensitive, or label sensitive. It
also save operator time and reduces reagent cost. [0064] c)
Eliminating the use of fluorescent labels, and being able to count
the cells in situ, removes the need for manual protocol steps such
as scraping cells off either side of the membrane in order to
separate the cells and count them. Using this invention, the assay
can now be run in an automated, homogeneous format. The only
existing homogeneous chemotaxis assay format, the Fluoroblok.TM.
system sold by Becton Dickenson, relies on using fluorescent labels
and is relatively expensive to comparable products. [0065] d)
Direct imaging of the cells on the surface of the membrane, without
using fluorescent probes or optical stains greatly enhances the
extension of these assays to a homogeneous, automated, multi-time
point data collection and analysis. Morphological changes to the
cells, such as shape change or actin polymerization can be
monitored in situ. In addition, direct cell imaging on the membrane
allows for other the measurement of individual cell-based
parameters not possible with current Boyden chamber solutions.
Individual cell migration and invasion assays parameters such as a)
speed and b) persistence of migration can be monitored kinetically.
[0066] e) Existing commercially available Boyden chamber solutions
dictate that at least 50,000 to 100,000 cells are required per
measurement well to achieve reasonable measurement precision. This
invention enables, for the first time, control of the pore size,
spacing and location. Using these design attributes, along with an
optimized upper reservoir capacity will allow for similar assay
precision using only a few thousand cells. [0067] f) Laser-based
photo-machining of the membrane allows precise control of both the
pore size, pore spacing and pore location, enabling, for the first
time, the ability to use these parameters to optimize the chemical
gradient formation process when used as a chemotaxis or
chemo-invasion measurement device. It is anticipated, that combing
this feature with a kinetic read-out of the cell migration process,
e.g. with an automated microscope such as the IncuCyte.TM. (Essen
BioScience, Ann Arbor Mich.), that the assay system will provide
more stable pharmacological data and reduce the artifactual effects
of agonist roll-over as depicted in FIGS. 7A and 7B, and the
potential confusion in measuring antagonist inhibition in these
types of assays.
REFERENCES
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L. Ajnderson, W. S. Ho, and W. J. Petzny, J., Model Pores of
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Schildhammer, Roman Zantl, Valentin Kahl, Elias Horn, "u-Slide
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