U.S. patent application number 12/407902 was filed with the patent office on 2010-09-23 for biochip assembly and assay method thereof.
Invention is credited to Dmitry Kashanin, Frank O'Dowd, Igor Shvets, Vivienne Williams.
Application Number | 20100240086 12/407902 |
Document ID | / |
Family ID | 42125968 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240086 |
Kind Code |
A1 |
Kashanin; Dmitry ; et
al. |
September 23, 2010 |
BIOCHIP ASSEMBLY AND ASSAY METHOD THEREOF
Abstract
The present invention is directed to a biochip assembly
comprising a semi-permeable membrane and an assay method using said
biochip assembly for carrying out cell based assays. Ideally, such
a method involves measuring the migration of cells in a channel
under the influence of an analyte wherein said cells are separated
from said analyte by a semi-permeable membrane and said analyte
and/or said cells are subjected to controlled flow conditions.
Inventors: |
Kashanin; Dmitry;
(Stepaside, IE) ; Shvets; Igor; (Castleknock,
IE) ; Williams; Vivienne; (East Wall, IE) ;
O'Dowd; Frank; (Ballybunion, IE) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
42125968 |
Appl. No.: |
12/407902 |
Filed: |
March 20, 2009 |
Current U.S.
Class: |
435/29 ;
435/288.3 |
Current CPC
Class: |
B01L 2300/0681 20130101;
G01N 33/5029 20130101; B01L 2400/0487 20130101; B01L 2300/0627
20130101; B01L 2400/0415 20130101; B01L 2200/14 20130101; B01L
2300/0864 20130101; B01L 3/502753 20130101; B01L 2300/0816
20130101; B01L 2300/16 20130101 |
Class at
Publication: |
435/29 ;
435/288.3 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method for measuring the migration of cells in a channel under
the influence of an analyte wherein said cells are separated from
said analyte by a semi-permeable membrane and said analyte and/or
said cells are subjected to controlled flow conditions.
2. The method of claim 1 wherein the method takes place in an
elongate enclosed channel having a semi-permeable membrane mounted
therein.
3. The method of claim 1 wherein said cells are present on at least
one side of said semi-permeable membrane and said analyte is
present on the opposed surface thereto.
4. The method of claim 1 wherein said sample cells and/or said
analyte are introduced into the channel at a controlled steady flow
rate.
5. The method of claim 1 wherein said cells or said analyte on one
side of said membrane is subjected to controlled flow.
6. The method of claim 1 wherein said cells or said analyte on both
sides of said membrane are subjected to controlled flow.
7. The method of claim 1 wherein said channel is a
microchannel.
8. The method of claim 1 wherein said analyte is a reagent liquid
or gel or a reagent eluting cell.
9. The method of claim 1 further comprising forming a layer of
seeded cells adjacent to at least one side of said semi-permeable
membrane.
10. The method of claim 9 wherein said layer of seeded cells is
formed and mounted in the channel prior to use.
11. The method of claim 1 further comprising coating at least one
side of the semi-permeable membrane with one or more substances
which effect cell function prior to forming a layer of seeded cells
on said semi-permeable membrane.
12. The method of claim 11 wherein the substance promotes adhesion
of cells.
13. The method of claim 11 wherein the substance is in the form of
a gel.
14. The method of claim 9 wherein the seeded cells form either a
confluent or non-confluent layer adjacent to said semi-permeable
membrane.
15. The method of claim 9 wherein the interaction between said
seeded cells and said sample cells is monitored and/or
recorded.
16. The method of claim 1 comprising a further step of coating the
internal bore of the channel prior to use with a substance which
interacts with said seeded and/or sample cells, preferably a cell
adhesion molecule and/or a cell transmigration substance.
17. The method of claim 16 wherein the physiological function of
said seeded cells is monitored and/or recorded.
18. The method of claim 9 wherein the physiological function of
said seeded cells is measured as a function of the shear stress
within the channel.
19. The method of claim 1 comprising introducing analyte to said
channel and monitoring and/or recording the response of said seeded
cells and/or sample cells to said analyte, in terms of adsorption,
metabolism and/or toxicity.
20. The method of claim 1 or claim 9 wherein cell to cell
interactions and cell to analyte interactions are measured.
21. The method of claim 1 wherein the flow is sustained by a
pressure driven pumping system or a positive displacement pumping
system.
22. A biochip assembly for carrying out assays with living cells
wherein the assembly comprises at least one elongate enclosed
channel having a semi-permeable membrane mounted therein.
23. The biochip assembly of claim 22 comprising a plurality of
elongate enclosed channels.
24. The biochip assembly according to claim 22 for carrying out
assays with living cells wherein the assembly comprises at least
one elongate enclosed channel having a semi-permeable membrane
mounted therein wherein the semi-permeable membrane acts as a
divider wall separating the elongate enclosed channel into a first
channel and a second channel
25. The biochip assembly of claim 24 wherein the first channel
receives sample cells and the second channel receives analyte or
vice versa.
26. A biochip assembly for carrying out assays with living cells
wherein the assembly comprises at least one elongate enclosed
channel having a semi-permeable membrane mounted therein wherein
the semi-permeable membrane forms a connecting wall between at
least two adjoining channels.
27. The assembly of claim 26 comprising a first and second channel
wherein the first channel receives said sample cells and the second
channel receives said analyte.
28. The assembly of claim 26 wherein the adjoining channels are in
line.
29. The assembly of claim 26 in which the adjoining channels
intersect at one section only.
30. The assembly of claim 24 wherein the semi-permeable membrane
abuts the elongate enclosed channel.
31. The assembly of claim 24 wherein the semi-permeable membrane
permits unidirectional or bidirectional flow.
32. The assembly of claim 24 wherein part of the channel is in
contact with said semi-permeable membrane.
33. The assembly of claim 24 wherein said semi-permeable membrane
comprises one or more semi-permeable membrane types characterized
by different membrane properties.
34. The assembly of claim 33 wherein each semi-permeable membrane
type has different pore size and/or different membrane size.
35. The assembly of claim 24 wherein at least one surface of the
cell transparent membrane comprises one or more substances which
effect cell function, preferably a substance which promotes the
adhesion of cells.
36. The assembly of claim 24 wherein the semi-permeable membrane is
a cell transparent membrane.
37. The assembly of claim 37 wherein the cell transparent membrane
is seeded with cells.
38. The assembly of claim 37 wherein the cell transparent membrane
is selectively permeable to different cell types.
39. A biochip assembly for carrying out assays with living cells
wherein the assembly comprises at least one elongate enclosed
channel and a well wherein a semi-permeable membrane separates said
channel from said well.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to a biochip assembly
comprising a semi-permeable membrane and a method using said
biochip assembly for carrying out cell based assays.
BACKGROUND OF THE INVENTION
[0002] The ability to monitor migration of biological cells through
tight layer of other cells and tissues is crucial for understanding
of mechanism of many life threatening diseases and development of
modern therapeutic drugs. This migration is typically triggered by
the presence of a particular chemical either immobilized on a
surface or diffused through a tissue.
[0003] In inflammatory conditions, for example, the migration of
leukocytes from blood vessels into diseased tissues is crucial to
the initiation of normal disease-fighting inflammatory responses.
At the same time, this process, known as leukocyte recruitment, is
also involved in the onset and progression of debilitating and
life-threatening inflammatory and autoimmunne diseases. Thus, the
ability to control the migration of leukocyte through blood vessels
into healthy tissues is an important pathway for development of
therapeutic treatment. This migration is complicated by the fact
that several leukocyte classes participate in this pathology
(including lymphocytes, monocytes, neutrophils, eosinophils and
mast cells) and each class carries out its own physiological
function. There is the whole range of chronic autoimmune diseases.
These include psoriasis, atherosclerosis, rheumatoid arthritis,
contact dermatitis, multiple sclerosis, inflammatory bowel disease,
hepatitis, sarcoidosis, idiopathic pulmonary fibrosis,
dermatomyositis and diabetes. There are also numerous organ
transplant rejection conditions such as allograft rejection and
graft-versus-host disease that are determined to a large extent by
the leukocyte migration.
[0004] The process by which leukocytes leave the bloodstream and
accumulate at inflammatory sites and initiate the disease takes
place in three distinct steps (Lawrence and Springer, 1991, Cell
65:859-73; Butcher E. C. 1991, Cell 67: 1033-36; Springer, T. A.
1990, Nature, 346: 425-33;). It is mediated by chemoattractant
receptors, by cell-surface proteins, called adhesion molecules, and
by the ligands that bind to these two classes of cell-surface
receptors. The major types of adhesion molecules are known as
"selecting" "integrins" and "immunoglobulin (Ig) family"
receptors.
[0005] Each of the three steps is essential for the migration of
the leukocytes to target tissues. Blocking these steps has been
shown to prevent a normal inflammatory response, and promotes
abnormal responses of inflammatory and autoimmune diseases (Harlan
et al., 1992, In vivo models of leukocyte adherence to endothelium.
In Adhesion: Its Role in Inflammatory Disease., J. M. Harlan and D.
Y. Liu, (eds.), W. H. Freeman & Co., pp. 117-150).
[0006] The three steps of leukocyte adhesion and transendothelial
migration can be summarized as follows:
[0007] Step 1--Primary adhesion. Leukocytes attach loosely to the
blood vessel endothelium and "roll" slowly along the blood vessel
wall, pushed by the flow of blood. Leukocyte-endothelium attachment
is mediated by cell surface adhesion molecules called "selecting"
which bind to carbohydrate-rich ligands ("glycoconjugates") on the
leukocyte cell surface.
[0008] Step 2--Activation of leukocytes and migration to the target
site. Chemoattractant receptors on the surface of the leukocytes
bind chemoattractants secreted by cells at the site of damage or
infection. Receptor binding activates the immune defenses of the
leukocytes, and activates the adhesiveness of the adhesion
molecules that mediate Step 3.
[0009] Step 3--Attachment and transendothelial migration. The
leukocytes bind very tightly to the endothelial wall of the blood
vessel and move to the junction between endothelial cells, where
they begin to squeeze between these cells to reach the target
tissue. This tighter binding is mediated by binding to adhesion
receptors called "integrins" on the leukocytes to complementary
receptors of the "Ig family" on the endothelium. (The Ig family
molecules are named for their similarity to antibody molecules
(immunoglobulins)). Chemoattractant receptors are also involved at
this stage, as the leukocytes migrate up a concentration gradient
of the chemoattractant secreted by cells at the target site.
[0010] It is increasingly clear that there may be another step
preceding primary adhesion, i.e. preceding step 1, called
"margination". As a result of margination leukocytes get pushed by
the red blood cells to the periphery of blood vessel, thereby
allowing leukocytes to interact with the endothelium. However,
margination is not commonly accepted as yet in the three step
migration process described above.
[0011] These three steps of receptor-ligand interactions are all
required and appear to act in a highly cooperative and coordinated
manner to mediate leukocyte adherence to the microvasculature,
diapedesis, and subsequent leukocyte mediated injury to tissue in
inflammatory disease.
[0012] LFA-1 and Mac-1 together comprise the leukocyte integrins, a
subfamily of integrins that share a common beta subunit (CD18) and
have distinct alphaL, alpha M and alpha.X (CD11a, b and c) alpha
subunits (Springer, 1990, Nature 346:425-433). They are required
for leukocyte emigration as demonstrated by an absence of
neutrophil extravasation (1) in patients with mutations in the
common beta subunit (leukocyte adhesion deficiency), and (2) after
treatment of healthy neutrophils with a monoclonal antibody (mAb)
to the common beta subunit in vivo or in vitro.
[0013] The integrins LFA (lymphocyte function-associated antigen)-1
and Mac-1 on the neutrophil bind to the Ig family member ICAM
(intercellular adhesion molecule)-1 on endothelium (Diamond et al.,
1990, J. Cell Biol. 111:3129-3139). LFA-1 binds to ICAM-2, an
endothelial cell molecule that is more closely related to ICAM-1
than these molecules are to other Ig superfamily members (Staunton
et al., 1989, Nature 339:61-64).
[0014] The integrin VLA-4, that contains the alpha.4 (CD49d)
subunit noncovalently associated with the betal (CD29) subunit, is
expressed by lymphocytes, monocytes, and neural crest-derived
cells, and can interact with vascular cell adhesion molecule-1
(VCAM-1) (Elices et al., 1990, Cell 60:577). Like ICAM-1 and
ICAM-2, VCAM-1 is a member of the Ig superfamily (Osborn et al.,
1989, Cell 59:1203).
[0015] Chemoattractants are soluble mediators which activate cell
adhesion and motility and direct cell migration through formation
of a chemical gradient. They are produced by bacteria and numerous
cell types including stimulated endothelial and stromal cells,
platelets, tumor cells, cultured cell lines, and leukocytes
themselves. The cells responding to chemoattractants appear to
express specific receptors on their surfaces which bind the
chemoattractant molecules and sense the gradient. Receptor
stimulation induces cells to respond via a common signal
transduction pathway which involves interaction of the
chemoattractant-receptor complex with a guanine nucleotide or
GTP-binding protein (G protein). This interaction stimulates
phosphatidyl inositol hydrolysis by a phospholipase C, thus
generating inositol phosphates and diacylglycerol. A transient rise
in cytosolic free calcium then activates protein kinase C, and a
variety of events including protein phosphorylation, membrane
potential changes, and intracellular pH alterations ensue.
[0016] Several of the chemoattractants primarily affecting
neutrophils were among the first chemoattractants identified. These
include the complement component C5a, arachidonate derivative
leukotriene B.sub.4 (LTB.sub.4), platelet activating factor (PAF),
and formylmethionyl peptides of bacterial origin such as
formyl-met-leu-phe (fMLP). Although structurally dissimilar and
stimulatory via separate receptors, these molecules produce a rapid
and marked increase in neutrophil adhesiveness and motility leading
to chemotaxis and prominent neutrophil accumulation in vivo. The
receptors for C5a and fMLP have been identified and sequenced; cDNA
clones for each have also been generated (Gerard and Gerard, 1991,
Nature 349:614-617). These receptors share many structural features
with one another and members of the "rhodopsin superfamily" of
protein receptors.
[0017] The chemoattractants which predominantly activate and guide
monocytes include monocyte chemoattractant protein-1 (MCP-1)
(Leonard and Yoshimura, 1990, Immunol. Today 11:97-101), the RANTES
protein (Schall et al., 1990, Nature 347:669-71), and the
neutrophil .alpha. granule protein CAP37, among others. MCP-1 and
RANTES are structurally homologous and belong to the subfamily of
chemoattractive cytokines that are defined by a configuration of
four cysteine residues in which the first two are adjacent (C--C).
CAP37's structure is most homologous to proteins of the serine
protease family (Peteira et al., 1990, J. Clin. Invest.
85:1468-76).
[0018] Compared with neutrophil and monocyte chemoattractants,
little is known about chemoattractants for lymphocytes. The best
characterized lymphocyte chemoattractants are RANTES and IL-8,
which primarily attract monocytes and neutrophils. Several in vitro
studies have described lymphocyte chemotactic activities in the
culture supernatants of mixed lymphocyte reactions and
mitogen-stimulated human peripheral blood mononuclear cells (Center
and Cruikshank, 1982, J. Immunol. 128:2563-68; Van Epps et al.,
1983, J. Immunol. 131:687).
[0019] Although considerable effort has been invested on the study
of lymphocyte chemoattractants, they remain poorly characterized
relative to monocyte and neutrophil chemoattractants.
Chemoattractants for the latter cell types, such as MCP-1 and IL-8,
have been purified based on the conventional chemotaxis assay,
sequenced, and cloned. However, no molecule identified primarily as
a lymphocyte chemoattractive factor has been sequenced and
cloned.
[0020] The devices for studies and monitoring of transmigration of
cells are well known in the fields of cell biology, life science,
medicine, pharmaceutical and the area of drug development. There
are also devices for filtering, growth and grouping of cells in
these fields.
[0021] The most widely used assay is the Boyden Chamber assay, in
which a microporous membrane divides two chambers, the lower
containing the test chemoattractant and the upper containing the
cells, e.g. lymphocytes. The microporous membrane is commonly
nitrocellulose or polycarbonate, and may be coated with a protein
such as collagen. The distance of migration into the filter, the
number of cells crossing the filter that remain adherent to the
undersurface, or the number of cells that accumulate in the lower
chamber may be counted.
[0022] Such a Boyden chamber is also known as a transwell chamber.
The chamber is made of well divided into two compartments, the
upper and lower chamber, by a filter containing pores. A
chemoattractant or other solution is placed in the lower chamber
and the suspension cell is placed in the upper chamber. Cells can
then migrate through the pores, across the thickness of the filter,
and toward the source of chemoattractant. Cells that migrated
across the filter and attached to the underside are then counted.
In some assays the membrane is coated with Extra-Cellular Matrix
(ECM) proteins (e.g. laminin, collagen, fibronectin) and then with
endothelial cells (EC). A variety of devices of this class as well
as the method for the transendothelial assay are described in U.S.
Pat. No. 5,514,555 (Springer).
[0023] Boyden chambers are commonly used for studies of disease and
also for the development of drugs for disease treatment. Here we
list some examples of these applications:
[0024] Prostate cancer: It is not fully understood at present time
the mechanism of the bone metastasis. However, interaction between
cancer cells and bone environment (extra cellular matrix: ECM)
seems critical for the process [Chen, N., et al., A Secreted
Isoform of ErbB3 Promotes Osteonectin Expression in Bone and
Enhances the Invasiveness of Prostate Cancer Cells. Cancer Res,
2007. 67(14): p. 6544-8]. The ability of prostate cancer cells to
penetrate a synthetic basement membrane was assessed in a
Matrigel-Boyden chamber invasion assay (BD Biosciences).
[0025] Allergy inflammation: the typical study is based on the
eosinophils migration. Assay performed in a 48-well microchamber
(neuroprobe) [Wong, C. K., P. F. Cheung, and C. W. Lam,
Leptin-mediated cytokine release and migration of eosinophils:
Implications for immunopathophysiology of allergic inflammation.
Eur J Immunol, 2007].
[0026] Migration of vascular smooth muscle cells: key step in
diseased arteries and may be controlled by ECM [Koyama, N., et al.,
Heparan sulfate proteoglycans mediate a potent inhibitory signal
for migration of vascular smooth muscle cells. Circ Res, 1998.
83(3): p. 305-13].
[0027] Chemotactic ability of dental plaque: Whole plaque
suspensions were chemotactic for polymorphonuclear leukocytes
[Miller, R. L., L. E. Folke, and C. R. Umana, Chemotactic ability
of dental plaque upon autologous or heterologous human
polymorphonuclear leukocytes. J Periodontol, 1975. 46(7): p.
409-14].
[0028] Boyden chambers/Transwell chamber and closely related
devices are available from a number of vendors such as BD
Biosciences; Corning; Neuroprobe; Millipore. Despite this, Boyden
Chamber assays are typically associated with certain shortcomings.
These include: [0029] Intravital microscopy studies have suggested
that leukocytes transmigration occurs over a time frame of minutes.
In contrast, the readouts of most Boyden chamber transfilter assays
are taken 1-4 hours after cell introduction. This excessive time
span is necessary in order to get reasonable statistics of cell
migration. [0030] No physiological flow can be established in this
assay thus is not possible to monitor cells though all stages of
leukocyte recruitment. [0031] There is no control of the gradient:
chemokine diffusion in the body might be different than in vitro as
it takes longer to get a cell migration on in vitro assays. [0032]
Changes in cell morphology during chemotaxis cannot be observed in
real-time (because cells transmigrate through the filter).
[0033] In addition, Boyden chamber assays cannot readily answer
many questions related to the leukocyte migration. This is
particularly true for the molecular and cellular mechanism of the
chemokine-induced transendothelial migration step of leukocytes
that still have not been fully elucidated. It is not clear at all
if positive, negative or any chemokine gradients are involved and
how such gradients may physically persist in relation to the
endothelium. Initially it was thought that chemokines form soluble
gradients across the blood vessel Endothelial Cells (ECs). However,
in blood even a short persistence of a soluble chemokine gradient
is not feasible because the constant flow of plasma removes the
soluble chemokines from the site of their production. Therefore, it
has been suggested already over a decade ago that only those
chemokines that have been physically retained (immobilized) on the
luminal membrane, for example by the glycosaminoglycan (GAG)
residues of glycoproteins, may be able to effectively induce the
integrin activation of the rolling leukocytes [Rot, A.,
Contribution of Duffy antigen to chemokine function. Cytokine
Growth Factor Rev, 2005. 16(6): p. 687-94]. It is known though that
a gradient of chemokine can direct cells and it is also well
established that ECs protein receptors are necessary for cell
adhesion.
[0034] There are other known assay types of assays for studies of
cell migration. For example, the Dunn chamber assay comprises
concentric rings separated by a bridge. The inner ring is filled
with medium and the outer ring is filled with chemoattractant
solution. Cells are cultured on a coverslip and placed upside down
onto the Dunn chamber. The assays allow observation of migrating of
cells towards the gradient formed between both rings.
[0035] Another area of applications that is broadly related to the
area of transmigration is the growth of mammalian cells. A number
of methods for culturing mammalian cells of different tissue
origins have been reported. However, many of these cells are
difficult to grow in vitro and, when grown, are not morphologically
similar to in vivo tissue. It would be desirable to produce a
tissue and cells which are morphologically similar to their in vivo
counterpart for in vitro toxicology and other studies (for example,
transepidermal drug transport).
[0036] Similarly there are requirements for tests of cells under
continuous flow conditions resulting from the area of toxicity.
Once identified, candidate drugs or modulators are usually
evaluated for bioavailability and toxicological effects (Lu, Basic
Toxicology, Fundamentals, Target Organs, and Risk Assessment,
Hemisphere Publishing Corp., Washington (1985); U.S. Pat. No.
5,196,313 to Culbreth and U.S. Pat. No. 5,567,592 to Benet).
Traditionally, early stages of drug discovery and development have
concentrated on optimizing binding and potency of experimental
compounds. Typically, animal studies are performed on late stage
pre-clinical drug candidates to characterize pharmacokinetics (PK),
pharmacodynamics (PD) and physiological toxicity. However, animal
studies are costly, time-consuming and are limited, by throughput,
to characterize no more than a few compounds. Furthermore, several
drugs have shown unanticipated or unpredicted side effects only
after reaching clinical trials or wide-scale release to the public.
The pharmaceutical industry has the ultimate goal of replacing
animal studies with in vitro tests that are validated, predictive
models for human toxicity and drug dynamics. More recently, the
industry has set a medium-term goal of creating high-throughput, in
vitro tests that annotate candidate compounds with adsorption,
metabolism and toxic (hereinafter referred to as "ADMET")
predictive parameters.
[0037] The toxicology of a candidate modulator can be established
by determining in vitro toxicity towards a cell line, such as a
mammalian, including human, cell lines. Candidate modulators can be
treated with, for example, tissue extracts, such as preparations of
liver (such as microsomal preparations) to determine increased or
decreased toxicological properties of the chemical after being
metabolized by a whole organism. The results of these types of
studies are often predictive of toxicological properties of
chemicals in animals, such as mammals, including humans. Current
methods designed to model drug absorption in vivo involve growing a
confluent layer of cells on a porous matrix that allows the test
compound to permeate through the cell layer and matrix to a bottom
well. It is desirable to carry out many of these measurements under
conditions of continuous flow. These would mimic better the real
physiological conditions. The complexity and interplay of
biological processes that must be simulated to predict the ADMET
properties of a compound far exceed the capabilities of currently
available methods and tools. For example, when a patient takes a
drug, it must first pass through the gastrointestinal tract and
penetrate into the bloodstream. The drug must then survive
oxidative modifications in the liver and get to the desired site
(e.g., target organ or primary tumor) in a sufficient therapeutic
concentration. Even if these biological functions could be
faithfully reproduced in vitro, a difficulty remains in getting the
capacity and format of the assay to facilitate testing and analysis
of thousands of compounds. Ideally, the assays should be versatile
enough to not only measure the enzyme cascade activity inside any
living or whole cell, no matter what its origin might be, including
cancer cells, tumor cells, immune cells, brain cells, cells of the
endocrine system, cells or cell lines from different organ systems,
biopsy samples etc., but should also be able to detect and measure
the permeability of the cell to the candidate compound, as well as
the metabolic activity of the cell on the candidate drug compound.
Methodologies are desired that will allow for the more rapid
acquisition of information about drug candidate interactions with
enzymes that may potentially metabolize the candidate drug, earlier
in the drug discovery process than presently feasible. This will
allow for the earlier elimination of unsuitable compounds and
chemical series from further development efforts, and also give an
investigator insight as to the nature of metabolites with potential
biological activity derived from the candidate drug. A parallel
flow chamber may be used for this purpose. However, there are
several disadvantages when using the parallel chamber. For example,
the parallel flow chamber requires a substantial amount of the drug
candidate for the experiment. Furthermore, setting up the
experiment is often time consuming and rather complex.
[0038] By way of example, liver hepatocytes express a family of
enzymes called cytochromes. One subfamily of cytochromes is known
as cytochrome P450. The cytochrome P450 enzyme (CYP450) family
comprises oxidase enzymes involved in the xenobiotic metabolism of
hydrophobic drugs, carcinogens, and other potentially toxic
compounds and metabolites circulating in blood. Efficient
metabolism of a candidate drug by a CYP450 enzyme may lead to poor
pharmacokinetic properties, while drug candidates that act as
potent inhibitors of a CYP450 enzyme can cause undesirable
drug-drug interactions when administered with another drug that
interacts with the same CYP450. See, e.g., Peck, C. C. et al,
Understanding Consequences of Concurrent Therapies, 269 JAMA
1550-52 (1993). Accordingly, early, reliable indication that a
candidate drug interacts with (i.e., is absorbed by, metabolized
by, or toxic to) hepatocytes expressing CYP450 may greatly shorten
the discovery cycle of pharmaceutical research and development, and
thus may reduce the time required to market a candidate drug.
Consequently, such earlier-available, reliable pharmacokinetic
information may result in greatly reduced drug development costs
and/or increased profits from earlier market entrance. Furthermore,
such earlier-available, reliable pharmacokinetic information may
allow a candidate drug to reach the public sooner, at lower costs
than otherwise feasible. Accordingly, extensive pharmacokinetic
studies of drug interactions in humans have recently become an
integral part of the pharmaceutical drug development and safety
assessment process, e.g., Parkinson, A., 24 Toxicological Pathology
45-57 (1996).
[0039] Thus, despite the advances made to date, there remains a
need to provide improved systems for carrying out cell based
assays.
SUMMARY OF THE INVENTION
[0040] The invention provides method and devices for performing
cell based assays and cell tests.
[0041] According to a first aspect of the invention, there is
provided a method for measuring the migration of cells in a channel
under the influence of an analyte wherein said cells are separated
from said analyte by a semi-permeable membrane and said analyte
and/or said cells are subjected to controlled flow conditions.
Essentially, the semi-permeable membrane is mounted in the channel
and acts as a divider wall defining a sample channel and an analyte
channel.
[0042] An objective of the invention is to provide a system and
method for the study of the migration of the cells under conditions
mimicking more closely in-vivo situation than some of the currently
available systems and methods.
[0043] A further objective is to provide a system and method for
the study of transmigration of the cells through a layer of cells
under conditions mimicking more closely the in-vivo situation, such
as for example the conditions of the continuous flow modeling shear
stress on cells, conditions of the pulsating flow modeling
conditions of pulsating shear stress.
[0044] Another objective is to provide a system and the method for
the study of cell-ligand interactions, such as the binding of cells
to ligands, and cell to cell interactions such as cell to cell
binding and adhesion.
[0045] A still further objective of the invention is to provide the
system for studies of the cell response or cell function to drug or
drug candidates. This response or function may include any of the
following by way of example. The test compound may: (1) kill or
decrease the viability of the test cell; (2) be metabolized or
chemically altered by the test cell; (3) pass through the test cell
unchanged, (4) be unreleasably absorbed by the test cell; (5) cause
the movement of the test cell through the membrane or substrate
surface; or (6) cause the detachment of the test cell from the
membrane or substrate surface.
[0046] The present invention aims to address at least some of the
above objectives.
[0047] Ideally, the migration of cells is transmigration and the
method of the invention facilitates the transmigration of cells
through the semi-permeable membrane.
[0048] Ideally, the sample cells and/or analyte are introduced at a
controlled steady flow rate across the channel/biochip. In this way
cells may be delivered across the semi-permeable membrane wherein
analyte is present on the opposed surface thereto.
[0049] Ideally, such a channel has a width in the range from
approximately 0.005 to 20 mm, more preferably in the range from
approximately 0.1 to 10 mm and a depth in the range of from
approximately 0.005 to 3 mm, more preferably in the range from 0.05
to 0.5 mm. According to one embodiment, the channel has a width of
approximately 100 to 500 microns. It will be understood that the
width and the depth of the channel do not need to be constant all
across its entire length, and indeed may change considerably
between different parts of the channel. The advantage of this is
that the assembly may be used to mimic situations where capillaries
or other portions of a patients body might be constricted. For
examples, blocking if the arteries and the like, may be easily
studied. The cross-sectional area or bore of the channel may be
cylindrical or non-cylindrical. Optionally, the bore size may be
chosen to mimic the bore size of capillaries or venules of a
human.
[0050] Ideally, the method takes place in an elongate enclosed
channel having a semi-permeable membrane mounted therein. It will
be understood the channel may be a microchannel, preferably an
elongate enclosed microchannel. In this manner, the semi-permeable
membrane may act as a divider wall in the elongate enclosed
channel, separating the sample channel from the analyte channel.
Alternatively, two or more elongate enclosed channels are connected
by the semi-permeable membrane,
[0051] Many different assays (e.g. monitoring cell transmigration
in a channel/biochip etc) may be carried out and examples of such
assays are expanded on below.
[0052] Preferably, said cells are present on at least one side of
said semi-permeable membrane and said analyte is present on the
opposed surface thereto.
[0053] In the method, the sample cells and/or the analyte are
ideally introduced into the channel at a controlled steady flow
rate. According to one embodiment, said cells or said analyte on
one side only of said membrane is subjected to controlled flow.
According to an alternative embodiment, said cells or said analyte
on both sides of said membrane are subjected to controlled
flow.
[0054] It will be understood that said analyte may be a reagent
liquid or gel. Thus, the analyte may be a chemoattractant, a toxic
substance and/or a pharmaceutical preparation. The reagent gel may
be in the form of a solid or semi-solid gel. For example, the
reagent liquid may comprise ECM gel containing IL-8. It will also
be understood that the reagent in certain cases may be a
placebo.
[0055] According to a preferred embodiment of this aspect of the
invention, the method further comprises forming a layer of seeded
cells adjacent to at least one side of said semi-permeable
membrane. Ideally, said layer of seeded cells is formed on the
semi-permeable membrane prior to use and said semi-permeable
membrane with seeded cells is mounted in the channel prior to use.
The seeded cells may form either a confluent or non-confluent layer
adjacent to at least side of said semi-permeable membrane. Such
seeded cells may be endothelial cells.
[0056] In another embodiment of this aspect of the invention, the
method further comprises coating at least one side of the
semi-permeable membrane with one or more substances which effect
cell function prior to forming a layer of seeded cells on said
semi-permeable membrane. Ideally, such substance promotes adhesion
of cells. Such substances may be in any form, such as a gel, liquid
etc.
[0057] The method may also comprise a further step of coating the
internal bore of the channel prior to use with a substance which
interacts with said seeded and/or sample cells. Such a substance is
ideally a cell adhesion molecule and/or a cell transmigration
substance. To facilitate the attachment of these substances, the
walls of the channel may be treated, e.g. by plasma treatment, so
that they become hydrophilic. Alternatively, they may be coated by
a hydrophilic coating such as liquid silicon. Such a hydrophobic
coating ensures that cells do not adhere to the walls of the
channel and detrimentally effect the results.
[0058] In one application of the method the interaction between
said seeded cells and said sample cells is monitored and/or
recorded.
[0059] In another application of the method, the physiological
function of said seeded cells is monitored and/or recorded.
[0060] In yet another application of the method, the physiological
function of said seeded cells is measured as a function of the
shear stress within the channel.
[0061] In a still another application of the method, the method
comprises introducing analyte to said channel and monitoring and/or
recording the response of said seeded cells and/or sample cells to
said analyte, in terms of adsorption, metabolism and/or
toxicity.
[0062] In another application, the method comprises measuring cell
to cell interactions and cell to analyte interactions.
[0063] It will be understood that the flow conditions may be
sustained by a pressure driven pumping system or a positive
displacement pumping system or any other suitable means.
[0064] In a preferred embodiment of the invention, the method
comprises causing sample cell containing liquid to flow in at least
one elongate enclosed channel having a semi-permeable membrane
mounted therein, thereby delivering sample cells against the
semi-permeable membrane having a reagent liquid on the opposed
surface thereto.
[0065] According to a second aspect of the invention, there is
provided a biochip assembly for carrying out assays with living
cells wherein the assembly comprises at least one elongate enclosed
channel having a semi-permeable membrane mounted therein.
[0066] Ideally, the assay is a transmigration assay and the method
involves measuring the transmigration of cells.
[0067] According to one embodiment, the assembly comprises a
plurality of elongate enclosed channels.
[0068] Ideally, the channel is a microchannel, preferably an
elongate enclosed microchannel.
[0069] Ideally, the semi-permeable membrane acts as a divider wall
separating the elongate enclosed channel into a first channel and a
second channel. In use, the first channel may receive sample cells
and the second channel may receive analyte or vice versa. It will
also be understood, that depending on the assay being carried out,
both channels may receive both cells and analyte.
[0070] According to another embodiment, the biochip assembly for
carrying out assays with living cells comprises at least one
elongate enclosed channel having a semi-permeable membrane mounted
therein wherein the semi-permeable membrane forms a connecting wall
between at least two adjoining channels. Ideally, the assembly
comprises a first and second channel wherein the first channel
receives said sample cells and the second channel receives said
analyte.
[0071] The channel assembly of the invention may be arranged in
several different ways. These type of constructions are described
in more detail in relation to the figures.
[0072] For example, the adjoining channels may be in line and as
such run parallel to each other. Alternatively, the adjoining
channels may intersect at one section of the channel only.
Additionally, the semi-permeable membrane abuts the elongate
enclosed channel.
[0073] The semi-permeable membrane may permit unidirectional or
bidirectional flow.
[0074] In one embodiment, only a defined part or length of the
channel is in contact with the said semi-permeable membrane.
[0075] In another embodiment, the semi-permeable membrane comprises
one or more semi-permeable membrane types characterized by
different membrane properties, for example each semi-permeable
membrane type has a different pore size and/or different membrane
size.
[0076] In yet another embodiment, at least one surface of the
semi-permeable membrane comprises one or more substances which
effect cell function, preferably a substance which promotes the
adhesion of cells. The semi-permeable membrane is ideally a
microporous membrane.
[0077] Ideally, the semi-permeable membrane is a cell transparent
membrane. Optionally, the cell transparent membrane may be seeded
with cells. The cell transparent membrane may be selectively
permeable to different cell types.
[0078] It is envisaged that the channel according to the invention
will generally have a planar top wall to allow good optical
properties for examination under a microscope and generally
speaking, the channel comprises planar top, bottom and side walls
(i.e. non-cylindrical cross-section).
[0079] It is also envisaged that assemblies comprising a plurality
of biochips as described above will be formed on one base sheet and
will preferably have various common feeder channels having ports
therein. This provides for ease of examination under the
microscope.
[0080] According to another aspect of the invention, there is a
biochip assembly for carrying out assays with living cells wherein
the assembly comprises at least one elongate enclosed channel and a
well wherein a semi-permeable membrane separates said channel from
said well. Ideally, the semi-permeable membrane is mounted in the
elongate enclosed channel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] The invention will now be more clearly understood with
reference to the following description given by way of example only
and the following non-limiting figures. For clarity in viewing the
drawings, where possible the same numbering for identical parts has
been used in FIGS. 1 to 15.
[0082] FIG. 1. is a plan view of a transmigration device of the
invention showing the sample channel and analyte channel in
line.
[0083] FIG. 2. is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing the semi-permeable membrane separating
the sample channel from the analyte channel.
[0084] FIG. 3. is a plan view of a transmigration device of the
invention showing the sample channel and analyte channel crossing
the analyte channel at one section comprising a semi-permeable
membrane.
[0085] FIG. 4. is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing two separate semi-permeable membranes
and a single compression gasket separating the sample channel from
the analyte channel.
[0086] FIG. 5. is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing a semi-permeable membrane separating
the sample channel from the analyte channel with two compression
gaskets.
[0087] FIG. 6. is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing the semi-permeable membrane separating
the sample channel from the analyte channel. In this embodiment one
of the channels depth is zero and the semi-permeable membrane abuts
one of the channels, preferably the analyte channel. The analyte
channel shown is coated with an ECM gel.
[0088] FIG. 7. is a plan view of a transmigration device of the
invention showing multiple sample channels intersected by a single
analyte channel.
[0089] FIG. 8. is a plan view of a transmigration device of the
invention showing a single sample channel intersecting multiple
analyte channels.
[0090] FIG. 9. is a plan view of a transmigration device of the
invention showing multiple sample channels intersecting multiple
analyte channels. Any number of channels may be used and the number
of analyte channels and sample channels may be the same or
different.
[0091] FIG. 10. is a plan view of a transmigration device of the
invention showing the sample channel and analyte channel in line
and two semi-permeable membrane types.
[0092] FIG. 11. is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing the semi-permeable membrane separating
the sample channel from the analyte channel. In this figure cells
are seeded on the semi-permeable membrane.
[0093] FIG. 12 is a side sectional view of the assembly along the
lines A-A' of FIG. 1 showing the semi-permeable membrane separating
the sample channel from the analyte channel. In this figure a
further layer of cells has seeded itself on the cells previously
seeded on the semi-permeable membrane.
[0094] FIG. 13 is side sectional view of an alternative embodiment
of the invention showing a single channel separated from a well by
a semi-permeable membrane.
[0095] FIG. 14 is an exploded perspective view of the construction
of the transmigration device showing the sample channel in line
with the analyte channel and where both channels are separated by a
semi-permeable membrane.
[0096] FIG. 15 is an exploded perspective view of the construction
of the transmigration device through the intersection of the
channels showing the sample channel crossing the analyte channel at
one section comprising a semi-permeable membrane.
DETAILED DESCRIPTION OF THE INVENTION
[0097] The invention provides a device and method for performing
cell-based assays and cell tests. Prior to discussing this
invention and figures in further detail, the following terms used
in the specification will first be explained.
[0098] The term "cell" includes both eukaryotic and prokaryotic
cells, including but not limited to bacteria, yeast, mammalian
cells. The use of plant cells may also be contemplated. Preferably
the cells are eukaryotic cells. According to one particularly
preferred embodiment the cells are leukocytes, such as neutrophils,
lymphocytes etc.
[0099] The term "sample cells" or "sample cell containing liquid"
ideally refers to a suspension of living cells within a suitable
carrier medium, for example, a culture medium. Such a culture
medium is ideally in liquid form but is not limited to this form.
It will be understood that more than one type of cell may be in the
suspension.
[0100] The semi-permeable membrane may be a cell-transparent
membrane. These terms will be used interchangeably in the
specification. The term "cell transparent membrane" encompasses a
film or membrane that contains holes or pores large enough that at
least some cells in the assays of interest can traverse into the
holes or pores and potentially migrate through the membrane.
Ideally, these holes or pores are large enough for a measurable
fraction of the cells to traverse. An example of Cell Transparent
Membrane is the membrane manufactured by Millipore Inc,
(www.millipore.com/cellbiology/cb3/microporousmembrane). Typically,
the membrane may be made of polymer film, e.g. polycarbonate,
hydrophilic PTFE, mixed cellulose, containing pores or holes of
defined range of dimensions. Ideally, the size of the holes in the
membranes may be in the range from approximately 1 .mu.m to 20
.mu.m in diameter. For certain cell types, it will be understood
that membranes containing holes of other dimensions could also be
used.
[0101] The membrane thickness is generally in the range from
approximately 1 .mu.m up to 1 mm, but more commonly is in the range
from approximately 10 .mu.m to 300 .mu.m. The Cell Transparent
Membrane is usually not hydraulically tight, meaning that it cannot
sustain a significant pressure difference between the two surfaces
of the membrane in a liquid-tight manner.
[0102] The term "seeded cells" covers cells which are grown on the
semi-permeable membrane. These seeded cells may form a confluent or
non-confluent layer. Ideally, endothelial cells may be used as the
seeded cells.
[0103] It will be understood that the semi-permeable membrane may
be coated with a substance which alters the seeded cell function or
promotes adhesion of the seeded cells to the semi-permeable
membrane prior to growth of the seeded cells. These substances may
include cell adhesion substances and/or cell transmigration
substances.
[0104] The term "reagent" or "reagent liquid"/"reagent gel" covers
many different types of analyte. The term "analyte" and "reagent"
may be used interchangeably in the specification. The reagent may
be a chemoattractant, a toxic substance and/or a pharmaceutical
preparation. For example, the reagent could be any liquid or gel
from a drug under assessment, a poison, a cell nutrient, a liquid
or gel containing other cells in suspension, reagent eluting cells
or indeed any reagent whose effect on the sample cells requires
assessment. It may also cover any reagent which activates a defined
cell function, such as a cell adhesion molecule or cell
transmigration molecule. In some embodiments of the invention, the
reagent may also be introduced into the sample cell channel. The
reagent gel may be in the form of a solid or semi-solid gel. For
example, the reagent liquid may comprise ECM gel containing IL-8.
It will also be understood that the reagent in certain cases may be
a placebo.
[0105] The term "cell function" means the biological or
physiological function of the cells such as cell mobility, cell
attachment, cell detachment, cell apoptosis, metabolism, cell death
due to the toxic effect of the environment, release of ligands,
release of agents involved in cell signaling, cell transmigration,
adsorption of the chemicals and ligands from the environment,
change in the cell shape, cell rolling and other broadly similar
functions.
[0106] The term "cell adhesion molecule" means any molecule which
facilitates cell adhesion. These include among others members of
the immunoglobulin superfamily (such as VCAM-1, ICAM-1, PECAM-1),
selectins (such as E-selectin, P-selectin, L-selectin), catherins
(such as E-catherin, N-catherin, P-catherin), integrins and any
other molecule which will facilitate adhesion of cells in the assay
to the walls of the device or the membrane. This also includes
components of the extracellular matrix such as collagen,
fibronectin, laminin. Any other suitable "cell adhesion molecules"
may also be contemplated.
[0107] The term "cell transmigration molecule" means any molecule
that will activate and facilitate the transmigration of the cells
in the assay. This includes chemokines (CC chemokines-such as
RANTES/CCL5, MCP-1/CCL2, CCL28; CXC chemokines-such as CXCL12; C
chemokines-such as XCL1; CX3C chemokines-such as
fractalkine/CX3CL1) and any natural or synthetic molecule that
induces a cell to migrate. Any other suitable "cell transmigration
molecules" may also be contemplated.
[0108] It will also be understood that the walls (i.e. the internal
bore) of the channels, in their entirety or part thereof, may be
coated with substances which interact with the sample cells. These
substances may include, but are not limited to enzymes, proteins,
polysaccharides, glycoproteins, both natural and synthetic
collagen. This may also include cell adhesion molecules and/or cell
transmigration molecules. To facilitate the attachment of these
substances to the walls of the fluidic channel it may be treated,
e.g. by plasma treatment, so that they become hydrophilic. This
type of treatment will be well known to those skilled in the
art.
[0109] The term "fluidic channel" or "elongate channel" covers a
channel wherein the length of the channel is greater than the
width/depth of the channel. Ideally, such a channel has a width in
the range from approximately 0.005 to 20 mm, more preferably in the
range from approximately 0.1 to 10 mm and a depth in the range of
from approximately 0.005 to 3 mm, more preferably in the range from
0.05 to 0.5 mm. It will be understood that the width and the depth
of the channel do not need to be constant all across its entire
length, and indeed may change considerably between different parts
of the channel.
[0110] The channel is typically made in polymer material but indeed
could also be made in glass or silicon or some other materials
either optically transparent or non-transparent.
[0111] Furthermore, for ease of manufacturing the cross-section
(internal bore) of the elongate channel is ideally non-cylindrical.
In most embodiments of this invention we will consider fluidic
channels of rectangular cross-section. These are just convenient
examples of fluidic channel cross-section that are easy to
fabricate and more easy to describe and are by no means limiting.
The fluidic channel cross-section could be of any other shape, e.g.
near rectangle with rounded corners, oval or semicircle, etc.
Ideally, the channel has a non-cylindrical cross-section.
Furthermore, the cross-sectional area of the channel may vary along
its length.
[0112] Ideally, the channel of the invention has an internal bore
of approximately 1 to 1000 micron in width, preferably 100 to 500
micron in width. Optionally, the bore of the channel may be
substantially identical to capillaries (e.g. of the order 8
microns), venules (e.g. of the order of 20 microns) or post
capillary venules of a human or other animals for example.
[0113] It will be understood that the channel may be a
"microfluidic channel" or "microchannel", such as an "elongate
enclosed microchannel". Optionally, the internal bore of the
microchannel, is substantially the same size as the post capillary
venules or capillaries of an animal, or more particularly, a human.
However, this is by no means limiting. It will be understood that
post capillary venules have an internal bore of below 50 micron in
width.
[0114] As it will be readily appreciated by those skilled in the
art, typically all the walls of a channel are liquid-tight. For
example in the case of a rectangular channel, the channel is
comprised of four walls, all of which are not transparent/permeable
to the liquid transported by it. In contrast, in our invention the
fluidic channel may be defined in a broader way, wherein in some
embodiments one or more walls of the fluidic channel can comprise a
Cell Transparent Membrane and therefore, the channel is not
necessarily liquid-tight. The channel does not need to be uniform
along its entire length. For example, only a fraction of the length
of the channel may comprise one wall comprising Cell Transparent
Membrane, while for the rest of the channel's length all of its
walls may be entirely liquid-tight. Furthermore, the channel may
contain membranes of several types. For example, one segment of the
channel may contain wall of membrane A and another segment of its
length may contain wall of membrane B having different
properties.
[0115] The term "flow inducing means" encompasses devices which
have the ability to induce flow. Generally this means devices
capable of supplying the pressure difference across fluidic
channel. This may be accomplished by means of syringes, various
types of pumps, including the pump as described in the U.S. Pat.
No. 6,805,841 (Shvets) which is incorporated herein by reference.
The flow can also be induced by means of electrophoretic or osmotic
pumping. In preferred embodiment the pressure is induced by means
of syringe pump or proprietary Mirus Nanopump.RTM. supplied by
Cellix Ltd (www.cellixltd.com).
[0116] The term "fluidic device" or "biochip assembly" means a
device comprising one or more channels or microchannels as defined
above being either mutually coupled to each other or decoupled, one
or more sample wells, one or more input ports and/or one of more
output ports and/or the coupling means such as tubing for coupling
liquids in and out from the channels.
[0117] In the context of the present invention generally, the
fluidic device is used for carrying out biological, medical,
chemical, biochemical, biotechnology or drug discovery experiments
or tests. The fluidic device may have one or more wells, sealed or
unsealed integrated with the device that may be coupled into the
channel(s) or decoupled from it (them). The fluidic device may be
substantially planar, that is built in into a substantially flat
substrate or non-planar. The fluidic device may operate with
external pumping means such as pump, syringe, pressure source,
electroosmotic pump. The fluidic device may have one or more
sensors integrated into it. The device may be connected to the
external or internal pumping means by means of rigid or flexible
tubing or any other suitable connection means. The channels may be
transparent to light or opaque. They can be made of polymer
material (e.g. PMMA, polystyrene, polycarbonate etc.), crystalline
material (e.g. Si), amorphous material (e.g. glass) or a
combination of several material types.
[0118] It will be understood that the term "transmigration" does
not necessarily imply that the sample cells must migrate all the
way across the membrane from the sample cell containing channel to
the reagent channel. The transmigration of sample cells across the
membrane is one of the most common assays that can be carried out
using the device. However, numerous other assays are also possible,
including but not limited to: [0119] seeding the sample cells on
the membrane, subjecting the sample cells immobilized on the
membrane to various chemical agents injected either into the sample
channel or the analyte channel, [0120] observation of the response
of the sample cells immobilized on the membrane to the toxic agents
injected at a known concentration, [0121] observation of the
detachment of the immobilized sample cells from the membrane back
into the sample channel, [0122] measurement of the shear force
causing the detachment of the immobilized sample cells from the
membrane, [0123] forming the layer of seeded cells on the membrane
and observation of interaction of the sample cells with the seeded
cells; [0124] migration of the sample cell through the layer of
endothelial cell grown on the membrane; [0125] observation of the
detachment of the sample cells from the cells seeded on the
membrane; [0126] observation of the attachment of the sample cells
to the cells seeded on the membrane.
[0127] Thus, the assay and method of the invention may be used in
the study of cell receptor-ligand interactions and cell-cell
adhesion followed by cell migration.
[0128] Referring to the drawings, one embodiment of the
transmigration device of the invention 1 is shown in FIG. 1. The
device 1 comprises a sample channel 3 and an analyte channel 6. In
this figure, the sample channel 3 and the analyte channel 6 are
in-line and ideally run parallel. In the embodiment shown in FIG. 1
each of the two channels has one input (2,5) and one output (4,7).
One could devise embodiments having more than one input and/or one
output. The two channels are separated by a semi-permeable membrane
8, ideally a cell transparent membrane, such as e.g. polycarbonate
membrane with pore size of 3, 5 or 8 um supplied by Millipore or
Nucleopore. In the overlap region 15 the two channels are separated
merely by a membrane 8. In the embodiment shown in FIG. 1 the
overlap region extends for most of length of the channels. One
could also devise an embodiment in which the overlap region covers
only a small segment of the channel's length.
[0129] It will be understood that the semi-permeable membrane 8 may
permit the passage of cells though it and as such may be a cell
transparent membrane. Passage of such cells may be unidirectional
or bidirectional.
[0130] It will be understood that the channel is subjected to
continuous flow (for example to mimic the flow of living cells
in-vivo). Ideally, both channels are subjected to continuous flow
in the same direction or in opposite directions. This assumes that
both channels contain sample cells and reagent (analyte) in the
form of liquid. If the reagent is in the form of a gel, the analyte
channel may be static.
[0131] FIG. 2 shows the cross-section A-A' of the two channels of
the transmigration device 1 of FIG. 1. Effectively the cell
transparent membrane 8 serves as the section of the common wall of
the two channels. The widths of the two channels (the sample
channel and the analyte channel) and their depths can be identical
or different. In some embodiments the channels are elongate
enclosed microchannels. FIG. 2 shows the two channels having the
same width but this represents only one particular example of the
transmigration device embodiment. Thus, the channels separated by a
semi-permeable membrane may have different widths.
[0132] In one embodiment, the sample channel 3 and the analyte
channel 6 can be made by imprinting of a flat plastic substrate
(e.g. ABS plastic, PEEK, PET, PMMA, polycarbonate, polypropylene
etc) by means such as hot embossing, injection molding or
lithographic pattern transfer technique. Ideally, the membrane 8 is
in the form of a sheet and is positioned between two substrates,
the sample substrate 9 and the analyte substrate 11 which contain
the sample channel 3 and the analyte channel 6 respectively. In
order to ensure a liquid tight connection, a compression gasket 13
of compressible polymer can be inserted in between the inner
surfaces of the two substrates (10, 12). FIG. 14 shows how this
particular construction may be assembled in practice.
[0133] It will be understood that the two channels, the sample
channel 3 and the analyte channel 6, do not have to be in-line or
parallel to each other, as shown in FIGS. 1, 2, 11 and 12. This is
shown schematically in the embodiment of the transmigration device
shown in FIG. 3. They can overlap, intersect or cross along an area
(the overlap region) that is small by comparison with the overall
area of the channel. FIG. 15 shows how this particular construction
may be assembled in practice.
[0134] Other embodiments of the invention can be contemplated,
including, embodiments having more than one membrane 8 or more than
one compression gasket 13. For example, FIG. 4 shows an arrangement
having two membranes, 16 and 17 and one compression gasket 13. In
this case the analyte could be positioned in between the two
membranes or alternatively two different types of cells could be
seeded on the membranes 16 and 17. Alternatively, the membranes 16
and 17 could be covered by two different types of enzymes. Still
alternatively, the membranes 16 and 17 could be membranes of
different properties for example different thickness or different
extent of transparency to the transmigrating cells. These
advantages of the embodiment comprising two membranes are given
here by way of example only.
[0135] Many other configurations of experiments are possible. FIG.
5 shows embodiment of the transmigration device with one membrane 8
and two compression gaskets 18 and 19. The advantage of this
embodiment could be that in some cases this transmigration device
could be easier manufactured as per embodiment utilizing two
compression gaskets and one membrane.
[0136] Many other embodiments could be readily devised by those
skilled in the art, some are expanded on below as non-limiting
examples of the invention.
[0137] For example, in some embodiments the membrane could be laid
against the flat inner surface of the substrate (e.g. sample
substrate or analyte substrate) and in others it could be bonded to
that surface e.g. by means of adhesive or ultrasonic welding. FIG.
6 shows an embodiment of the transmigration device in which the
depth of the analyte channel is zero. In this embodiment the
semi-permeable membrane 8 abuts either one of the channels. In this
embodiment the analyte substrate is a flat substrate. The analyte
substrate may be provided with a gel containing, for example, a
cell transmigration reagent such as ECM gel 20. Thus, a layer of
gel may be sandwiched between the semi-permeable membrane and the
analyte substrate.
[0138] With reference to FIG. 1, the sample channel input 2 and/or
the analyte channel input 5 may be connected to a pumping means to
provide cell flow and shear stress. In addition, the sample channel
output 4 and/or the analyte channel output 7 may be connected to
the collection means such as set of wells or collection reservoir.
These are not shown in FIG. 1 for brevity but will be well known to
those skilled in this field and are described in the following U.S.
Pat. Nos. 6,770,434, 6,805,841 and 7,122,301 and U.S. patent
application Ser. No. 10/500,277 by the same inventors the contents
of which are herein incorporated by reference.
[0139] The following describes one example of the operation of the
device. The width of both, the sample channel 3 and the analyte
channel 6 is ideally approximately 400 .mu.m and the channel depth
is ideally approximately 100 .mu.m. The analyte channel is filled
with an ECM gel containing approximately 1-10 nM of IL-8 at the
temperature of approximately 4 degrees C. The gel in the analyte
channel is allowed to solidify at the temperature of 37 degrees C.
for 30 minutes. Primary neutrophil cells were isolated from the
whole blood according to the protocol known to those skilled in the
field. The neutrophil cells were re-suspended in a culture medium,
for example RPMI1640 (Gibco) at the concentration of 5 million/ml
and injected into the sample channel and flow at the average linear
velocity of 0.83 mm/sec corresponding to the shear stress in the
range of 0.5 dyne/cm.sup.2. The sample cells 14 are schematically
shown in FIG. 2. The flow/continuous flow can be supported by means
of a suitable pump such as e.g. Mirus Nanopump.TM. from Cellix Ltd.
The duration of the experiment may be in the range of 20 minutes to
2 hours, although other durations may of course be contemplated.
For the duration of the assay, transmigrating and resting
neutrophil cells are supplied with a culture medium (necessary to
ensure cell viability and survival) through the sample channel 3.
The cells migrating through the membrane are ideally observed by
means of optical microscope and the number of cells migrating
through the membrane is monitored by means of automatic image
recognition software such as DucoCelI.TM. analysis software
supplied by Cellix Ltd. The results of the experiment can then be
compared with reference results obtained under similar conditions,
i.e. with the difference being that the ECM gel in the analyte
channel does not contain IL-8.
[0140] Although, it is contemplated that a first channel of the
device contains the sample cells (the "sample channel") and a
second channel comprises the reagent (the "analyte channel"), the
channels may also comprise additional substances depending on the
type of assay being carried out. For example, in order to ensure
the sample cells are viable, it may be necessary to introduce a
cell culture medium to the channel. Alternatively, liquid media may
be needed to supply the cells with oxygen. Furthermore, it may be
necessary to stain one or both of the channels. Additionally, the
reagent may be introduced into the channel mixed with a gel. Such
gels may initially be in the form of a liquid that turns into a gel
as the temperature changes. Still additionally, one may want to
test a drug candidate to assess changes to transmigration through
the membrane caused by a chemoattractant.
[0141] In a preferred embodiment of the invention, sample cells are
introduced into one channel (e.g. first/sample channel) and a
chemical agent that effects their migration is introduced into a
second channel (e.g. analyte channel). Such a chemical agent may be
in the form of a gel, so that the second channel/analyte channel is
static. Optionally, the semi-permeable membrane may have a layer of
cells seeded on it, enabling the study transmigration of cells
through the seeded cell layer.
[0142] In an alternative embodiment of the invention, sample cells
A may be introduced into a first channel and reagent eluting sample
cells B may be introduced into a second channel. Each channel may
also contain cell growth media. 2. Sample cells B may then release
a reagent that causes cells A to migrate from the first channel to
the second channel. It will be understood that many other
configurations will be possible.
[0143] Further embodiments of the transmigration device are shown
in FIG. 7, 8 and 9. These embodiments comprise multiple
intersections of sample and analyte channels. For example, FIG. 7
shows the embodiment where one analyte channel 22 (analyte channel
input 21 and analyte channel output 23) crosses a number of sample
channels 25a to 25e (sample channel input 24a to 24e). The
locations of intersections of the analyte channel 22 with the
sample channels 25a to 25e include membranes as described with
reference to FIGS. 1 to 6. The membranes are not shown in FIG. 7.
It will be understood that the widths of all the sample channels do
not have to be identical. The sample channels can transport
different cell types (C.sub.1, C.sub.2, . . . , C.sub.n) and in
this way the transmigration function could be tested in a variety
of cell lines at the same time against the same cell transmigration
molecule. The sample channel outputs are not shown in FIG. 7.
[0144] Likewise the same sample channel 27 (sample channel input
26) could intersect a number of analyte channels 29a to 29d
(analyte channel input 28a to 28d, analyte channel output 30a to
30d) and this embodiment is shown in FIG. 8. In this way the same
cell type could be tested against the variety of cell
transmigration molecules. The sample channel output is not shown in
FIG. 8.
[0145] For example, consider FIG. 8, with the liquid in the sample
channel 27 moving downwards. Then there is increasing concentration
of the same chemical in the analyte channels subsequent 1,2,3,4
marked with numerals 29a, 29b, 29c, 29d, respectively, e.g. analyte
channel 1 (29a) has concentration x, channel 2 (29b) : 5x, channel
3 (29c): 25x, channel 4 (29d): 125x. Then the contamination from
the analyte channel 1 (29a) should be negligible as at the point of
the analyte channel 2 (29b) intersection with the sample channel,
the concentration of the same analyte is much higher anyway.
Likewise the concentration of the analyte from the analyte channel
2 (29b) at the cross section of analyte channel 3 (29c) with the
sample channel is also much smaller than the concentration of the
analyte directly from the analyte channel 3 (29c), etc. In this
manner cross-contamination is not an issue. In addition, if the
reagent in the analyte channel is in the form of a gel,
cross-contamination is not an issue as the gel would not be
expected to elute much reagent into the sample channel.
[0146] In addition, another embodiment may comprise the
intersection of a variety of sample channels 32a to 32e and analyte
channels 34a to 34f in one transmigration device 1 as schematically
shown in FIG. 9. In this way the variety of cells could be
simultaneously tested against the variety of cell transmigration
molecules. The sample channels outputs are not shown in FIG. 9.
Sample channel inputs 31a to 31e are shown along with analyte
channel inputs 33a to 33f and analyte channel outputs 35a to
35f.
[0147] FIG. 10 shows an embodiment of the transmigration device
whereby the cell transparent membrane has several regions 36 and 37
each one being characterized by a different set of membrane
properties. For example, the membrane could comprise two areas each
one being characterized by a different pore size. Alternatively, it
could be comprised of two areas having the same pore sizes but
different membrane thickness. These regions are schematically
marked in FIG. 10 as overlap region 1 (38) and overlap region 2
(39). It will be understood that a single cell transparent membrane
may have different membrane properties or when multiple membranes
are used, each separate membrane may have different properties. If
multiple membranes are used, they may form a continuous membrane
area or a non-continuous membrane area.
[0148] FIG. 11 shows another embodiment of the transmigration
device where a layer of cells 40 is allowed to form on the
membrane. The layer of cells could be e.g. a confluent layer of
endothelial cells. However, other cells could also be seeded on the
membrane. Thus, in one embodiment, the layer is a confluent layer
of cells.
[0149] Alternatively, the layer of cells are non-confluent i.e.
bare areas could be left on the membrane 8.
[0150] To seed the cells on the membrane 8, the membrane could be
covered by specific adhesion molecules such as fibronectin (Sigma
Inc). More preferably the layer of cells is grown on the membrane
when it is removed from the transmigration device and placed into
the cell culture incubator at 37.degree. C., 5% CO.sub.2 and 80%
humidity. Typically endothelial cells are seeded at the density of
approximately 75000/cm.sup.2 and allowed to grow for a period of 48
hours until a confluent layer of endothelial cell is formed.
Different primary endothelial cells and cell lines may require
different density and seeding time. Following seeding, the membrane
with seeded cells 40 is placed between the sample and the analyte
channels 3 and 6 and sealed by way of compression gasket 13.
Subsequently, sample cells 14 are injected into the sample channel
3 and the interaction between the sample cells 14 and the seeded
cells 40 is observed. This interaction could include by way of
example the migration of the sample cells through the layer of
seeded cells, migration of the sample cells through the layer of
seeded cells and thought the membrane, adhesion of the sample cells
to the seeded cells.
[0151] In a further embodiment, the layer of the sample cells 14
could seed on the layer of the seeded cells 40 as shown in FIG. 12.
Then the sample cells 14 could be subjected to interaction with
various chemicals injected either in the sample channel or the
analyte channel. In yet a further embodiment the layer of seeded
cells as shown in FIG. 12 could be subjected to the chemical
injected either in the sample channel 3 or in the analyte channel
6.
[0152] FIG. 13 shows an alternative embodiment of the device of the
invention in which one of the channels is a well 42. Ideally, the
analyte channel is in the form of an open well 42, with no input or
output. In this embodiment, transmigration molecules may be in the
form of a gel, which is placed in the well 42. The semi-permeable
membrane is placed at the bottom of the gel.
[0153] FIGS. 14 and 15 show expanded perspective views of the
contraction of the devices of FIG. 1 and FIG. 3 respectively. These
figures show how the biochips 1 are made of sheets of plastic
materials bonded together. Again, parts similar to those described,
with reference to the previous drawings, are identified by the same
reference numerals. FIG. 14 shows the channels (non-cylindrical
bore) 3 and 6 formed in plastics material surrounding a sheet of
membrane 8. The channels are in-line . FIG. 15 shows the channels 3
and 6 separated by the sheet of membrane 8 crossing at a single
intersection location.
[0154] Cells flowing through the channels may be observed via a
microscope and images may be captured and analysed at a later date.
For this one could use conventional microscope or alternatively, a
more specialist miscroscope, such as confocal or fluoresecent
microspe or indeed any other microscope known in the field of cell
imaging and analysis.
[0155] Various different imaging technologies can be used in
conjunction with the invention. Furthermore, various different
image processing programmes for analysis and processing of the
acquired images can also be used in conjunction with the invention.
Finally, one could use positive displacement or pressure driven
pump to move cells and analyte in the channels.
[0156] Some examples of these technologies and software follow.
[0157] For example, Cellix Ltd. has developed a novel Microfluidic
Platform consisting of a PC-controlled Nanopump.RTM. with
microfluidic biochips (such as Vena8.RTM. biochips (Cellix Ltd.)
previously developed or the elongate enclosed channels of the
invention) and DucoCell.RTM. (Cellix Limited) analysis software.
The Nanopump.RTM. enables very accurate flow rates to be achieved
which are more reproducible and consistent compared to anything
currently available. Importantly, flow rates are extremely low (5
pL min-1 to 10 pL min-1) and the shear stress levels that the pump
can mimic (up to 30 dyne cm-2) are equivalent to those found in
blood vessels in vivo. The Nanopump.RTM. is vital to the use of
small diameter capillaries as standard syringe pumps are incapable
of delivering the required low flow rates.
[0158] In order to carry out an assay using the above platforms,
the following general protocol may be used: [0159] First of all,
the cell type to be analysed must be determined, followed by
establishing how to harvest such cells e.g. culturing in growth
media, or isolation from in vivo fluids. [0160] Secondly, the assay
itself should be outlined, including whether live cells or proteins
will be coating the channels of the biochip. If it is the former,
protocols for culturing the cells both outside and inside the
biochip channel must be established. Thirdly, the adhesion profile
of the cells to be passed through the coated channel should be
determined. [0161] Next, if exogenous compounds are being analysed,
these should then be introduced to the system and their effect on
the adhesion profile assessed.
[0162] This should include calculation of required concentrations
and pre-incubation conditions, before introduction to the system.
Finally, the images taken via the digital camera attached to the
microscope should be masked and analysed using the Ducocell.RTM.
software.
[0163] Various specific assays may be contemplated using these
platforms including but not limited to: [0164] A microfluidic assay
assessing the effect of levocetirizine on human eosinophil
adhesion, involving the following [0165] The method involves
coating each microcapillary for one hour in humid conditions at
ambient temperature with either human vascular cell adhesion
molecule-1 (rhVCAM-1) or bovine serum albumin (BSA) (both 10 .mu.g
mL-1 in HBSS containing Ca2+ and Mg2+). All capillaries were then
coated with BSA to occupy non-specific binding sites. Resting or
Granulocyte-macrophage colony-stimulating factor (GM-CSF)--treated
eosinophils were pre-incubated at 37.degree. C. in a water bath for
10 mins before incubation with/without levocetirizine (0.1 nM-100
nM), with anti-VLA-4 mAb as a positive control) for a further 20
mins. [0166] Eosinophils were infused into the capillaries
(microfluidic biochips) at stepwise increases in shear stress, from
0 to 5 dyne cm-2, one minute per shear stress level. Images at each
shear stress level were captured using the accompanying PixeLINK
microscopy software. For experiments with GM-CSF-stimulated (1 ng
mL-1) eosinophils, the cytokine was added to the warmed cells at
the same time as levocetirizine and incubated at 37.degree. C. for
20 mins prior to commencing the flow assay. Adhesion was evaluated
by monitoring eosinophil migratory behaviour in real time with
images captured via a digital camera connected to the microscope.
[0167] Image analysis--several images per shear stress level may be
captured and adhered eosinophil numbers can be recorded using
Ducocell.RTM. application software. Data was exported into Excel
for interpretation. Statistical significance was determined by
Students unpaired t-test, and P>0.05 was considered
statistically significant. Data was presented as mean.+-.s.e.mean.
[0168] A microfluidic assay assessing the adhesion profiles of
peripheral blood lymphocytes (PBLs). [0169] A microfluidic assay
assessing the adhesion profile of platelets on various matrix
proteins. [0170] A microfluidic assay assessing novel
anti-inflammatory effects of montelukast (MLK) on resting and
GM-CSF-stimulated eosinophils using the Cellix VenaFlux.RTM.
platform to mimic physiological adhesion to rhVCAM-1. [0171] A
microfluidic investigation of T-cell adhesion to ICAM-1 with a
mixed sepsis model treated with a range of statins under
physiological shear stress using the Cellix Microfluidic Platform
SP 1.0. [0172] Cell Harvesting and Sample Treatments [0173]
Peripheral blood was donated by 6 healthy subjects. Following
mononuclear cell isolation, monocytes were allowed to adhere to the
culture vessels before B-cells were removed from the T-cell
population using nylon wool adhesion. [0174] T-cells were then
co-cultured in the presence of monocytes. [0175] Cells were treated
with 10 nM meva-, lova- or simvastatin dissolved in ethanol (0.1%
v/v final) or prava- or fluvastatin dissolved in water. Cells were
then stimulated with 2 .mu.g/ml lipopolysachharide (LPS) and 20
.mu.g/ml peptidoglycan G (PepG). Control cells were treated with
0.1% v/v ethanol.+-.LPS/PepG and incubated a humidified 37.degree.
C. incubator containing 5% CO2 for 18 hours. [0176] Biochip Coating
Procedures [0177] Each microchannel (400 .mu.m wide, 100 .mu.m
deep) was coated overnight in humid conditions at 4.degree. C. with
rhICAM-1 (10 .mu.g/ml), before being coated with BSA, 10 .mu.g/ml.
Two additional channels were coated with BSA for 2 hrs at room
temperature. Prior to shear experiments, all channels were washed
thrice with media. [0178] Adhesion Profiles [0179] Isolated T cells
were infused into the rhICAM-1 and BSA coated channels under a
defined shear stress of 0.5 dyne cm-2 for a time period of 5
minutes in CO2 independent media. [0180] Images were captured using
the accompanying PixelLink imaging software. [0181] Image Analysis
[0182] T cell adhesion profiles of single cells were recorded using
DucoCell.RTM. software. Cell images were captured from three
microscopic fields from each channel. Data was exported into Excel
to allow further analysis. [0183] Statistics [0184] Data obtained
from this experiment can be analyzed using the Wilcoxon's
signed-rank test using Graphpad Prism.RTM. 4 software. [0185] A
microfluidic assay to elucidate the importance of physiological
shear stress environment required for E. coli adhesion,
colonisation and biofilm formation using the Cellix VenaFlux.RTM.
Platform. [0186] Biochip Coating Procedure: [0187] Each
microchannel (400 .mu.m wide, 100 .mu.m deep) was coated in humid
conditions at 37.degree. C. for 45 min with 10 .mu.l of either 200
.mu.g/ml mannose-BSA (monomannase), 20 .mu.g/ml RnaseB
(trimannose); or 10% BSA. Prior to flow experiments each channel
was washed three times with PBS-0.2% BSA and quenched with PBS-0.2%
BSA for 15 min to decrease non-specific binding. [0188] Adhesion
Profiles: [0189] Bacteria was infused into the 1M, 3M and BSA
coated channels using pre-defined shear steps from 0.1, 0.3, 1.0
and 8.0 dyne/cm.sup.2, 100 s per shear stress level. [0190] Image
Analysis: [0191] E. coli adhesion profiles of single cells were
recorded using MetaMorph software. Cell images were captured from
three microscopic fields from each channel and further analysed by
Image J and Cellix's DucoCell.RTM. software. Data was exported into
Excel to allow further analysis. [0192] A microfluidic assay to
examine differential cell adhesion within an isogenic model of
melanoma progression under physiological shear flow conditions
using the Cellix VenaFlux.TM. Platform. [0193] A microfluidic assay
to screen a range of novel thermoresponsive polymers designed to be
used as dual drug-eluting systems in coating stents. Specifically,
to assess the ability of xemilofiban released in conjunction with
fluvastatin, to prevent thrombus formation/platelet adhesion to
fibrinogen using Cellix's VenaFlux.RTM. platform.
[0194] It will be understood that the device described may be
called a transmigration device. As explained above, this term does
not imply that the sample cells must migrate all the way across the
membrane from the sample channel into the analyte channel. Such a
transmigration of sample cells across the membrane is indeed one of
the most common assays that can be carried out using the device.
However, we also described numerous other assays such as seeding
the sample cells on the membrane, subjecting the sample cells
immobilized on the membrane to various chemical agents injected
either into the sample channel or the analyte channel, observation
of the response of the sample cells immobilized on the membrane to
the toxic agents injected at a known concentration, observation of
the detachment of the immobilized sample cells from the membrane
back into the sample channel, measurement of the shear force
causing the detachment of the immobilized sample cells from the
membrane, forming the layer of seeded cells on the membrane and
observation of interaction of the sample cells with the seeded
cells; migration of the sample cell through the layer of
endothelial cell grown on the membrane; observation of the
detachment of the sample cells from the cells seeded on the
membrane; observation of the attachment of the sample cells to the
cells seeded on the membrane. Thus, the device of the invention may
be used to carry out multiple assays on living cells, the
transmigration aspect is merely a preferred embodiment of the
invention.
[0195] In the specification, the terms "comprise, comprises,
comprised and comprising" or any variation thereof and the terms
"include, includes, included and including" or any variation
thereof are considered to be totally interchangeable and they
should all be afforded the widest possible interpretation.
[0196] The invention is not limited to the embodiment hereinbefore
described, but may be varied in both construction and detail within
the scope of the claims.
* * * * *