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Author Manuscript
Lab Chip. Author manuscript; available in PMC 2011 June 21.
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Published in final edited form as:
Lab Chip. 2010 June 21; 10(12): 1596–1603. doi:10.1039/b927316f.
Microdevice to capture colon crypts for in vitro studies
Yuli Wanga, Rahul Dhopeshwarkara, Rani Najdib, Marian Watermanb, Christopher E. Simsa,
and Nancy Allbrittona,c
a Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599
b
Department of Microbiology & Molecular Genetics, University of California, Irvine, California,
92697
c
Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC
27599 and North Carolina State University, Raleigh, NC 27695
Abstract
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There is a need in biological research for tools designed to manipulate the environment
surrounding microscopic regions of tissue. In the current work, a device for the oriented capture of
an important and under-studied tissue, the colon crypt, has been designed and tested. The objective
of this work is to create a BioMEMs device for biological assays of living colonic crypts. The end
goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a
manner that recapitulates the in vivo state. Crypt surrogates, polymeric structures of similar
dimensions and shape to isolated colon crypts, were used in the initial design and testing of the
device. Successful capture of crypt surrogates was accomplished on a simple device composed of
an array of micron-scale capture sites that enabled individual structures to be captured with high
efficiency (92 ± 3%) in an ordered and properly oriented fashion. The device was then evaluated
using colon crypts isolated from a murine animal model. The capture efficiency attained using the
fixed biologic sample was 37 ± 5% due to the increased variability of the colon crypts compared
with the surrogate structures, yet 94% ± 3% of the captured crypts were properly oriented. A
simple approach to plug the remaining capture sites in the array was performed using inert glass
beads. Blockage of unfilled capture sites is an important feature to establish a chemical gradient
across the arrayed crypts. A chemical concentration gradient (Cluminal/Cbasal > 10) was
demonstrated across the arrayed crypts for over 8 h. Finally unfixed colon crypts were
demonstrated to be effectively captured by the micromesh array and to remain viable on the
capture sites at 5 h after mouse sacrifice. The present study demonstrates the feasibility and
potential for rationally microengineered technologies to address the specific needs of the biologic
researcher.
Introduction
The ability to monitor and control the microenvironment of cells and tissues is one of the
most promising applications for microengineered systems.1 Microfluidic devices have made
it possible to easily and accurately modify the fluidic microenvironment of cells both
temporally and spatially. For example, exposure of cells to chemotactic gradients in a
quantitative manner can now be performed routinely through the use of microfluidic
gradient makers.2, 3 Similarly, spatially discrete stimuli can be applied even to a portion of
a single cell in order to study molecular responses at the subcellular level.4 While much of
the effort to control the environment on the micron scale has been directed at single cells,
many areas of biological research would benefit from devices designed to manipulate the
nlallbri@unc.edu; Fax: +1 (919) 962-2388; Tel: +1 (919) 966-2291.
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environment surrounding microscopic regions of tissue. Such devices would enable
biologists to more accurately recapitulate the stem cell niche, to influence the tumor
microenvironment, and to manipulate inflammatory infiltrates, all of which are high priority
areas of investigation.
Since many tissues are polarized with opposing sides exposed to different fluidic
microenvironments, particularly intriguing is the potential for manipulating these
environments in a discrete and independent manner. One area of research that would benefit
from this selective control is the study of colon physiology in health and disease. The large
intestine, or colon, is the last portion of the digestive system in most vertebrates.5 The
principle functions of the colon are the re-absorption of salts and water and the elimination
of undigested foodstuffs and other wastes.6 The surface of the colon is made up of a single
layer of columnar epithelial cells, which form tube-like invaginations into the underlying
connective tissue to form the basic functional unit of the colon, the crypt.7 The normal colon
consists of millions of crypts with each crypt containing about 2,000 cells.8–10 The luminal
face of the crypt is continually exposed to colonic contents and serves as a barrier to bacteria
while simultaneously enabling absorption of water and electrolytes needed to maintain
homeostasis. The opposing face of this epithelium lies adjacent to connective tissue and
blood- or lymph-filled capillaries that serve to support the structural and metabolic needs of
the colonic mucosa.
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The colonic mucosa is one of the most vigorously self-renewing tissues of adult mammals.
11 The proliferative cells that serve to replenish the mucosa reside at the base of the crypts
where stem cells give rise to progeny that terminally differentiate into colonic epithelial cells
as they transit the crypt. Colon cancer, one of the most common cancers to afflict mankind,
is thought to arise from the stem cell or its progeny, and study of the stem cell niche in the
colonic crypt is an active area of research.7, 12, 13 Inflammatory bowel diseases,
autoimmune diseases that include ulcerative colitis and Crohn’s disease, cause significant
human suffering.14 These diseases damage the colon through attack of crypt cells by
inflammatory infiltrates. Due to technical challenges in the in vitro assessment of isolated
crypts, studies of the various diseases involving colonic crypts have been restricted primarily
to in vivo inspection and histological evaluation.7, 15 In vivo studies by endoscopy or
noninvasive imaging enable examination of living colonic tissue at a macroscopic, but not
cellular scale. Histologic evaluation of fixed tissue enables study at the cellular level, but the
rich and dynamic qualities of the crypt tissue are lost.
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There are many areas of investigation that would benefit from the ability to study the
isolated colon crypt temporally while manipulating the environments of its luminal and
basal sides. With the recent discovery of the colon stem cell marker lgr5, the impact of
various environmental perturbations, including toxins or growth factors, on the stem cell
niche could be directly studied.11, 16 The role of dietary components on crypt physiology
could be readily tested, for example the loading of dietary calcium and its promotion of
apoptosis as a mechanism for cancer prevention.17 Selective exposure of luminal and basal
aspects of the crypt to chemotactic agents and other inflammatory mediators would facilitate
the study of the interplay of inflammation and colonic epithelium in real time. With recent
advances in the long-term culture of isolated colon crypts from mouse model systems,
technologies to enable controlled, in-depth studies of this tissue are needed.12, 18 In the
current study, the preliminary work to build a microdevice for the in vitro study of isolated
mouse colon crypts at the interface of two different environments is presented. Several
difficulties have been overcome to address the task of capturing isolated three-dimensional
structures in a selective orientation that will allow the luminal and basal sides of the crypts
to be differentially exposed. Microdevices to capture isolated tissue possessing a complex
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shape for in vitro study, such as that presented here, have enormous potential in future
biomedical research.
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Materials and Methods
Materials
SU-8 photoresist (formulation 10) was purchased from MicroChem Corp. (Newton, MA).
The Sylgard 184 silicone elastomer kit was purchased from Dow Corning (Midland MI).
Gamma-butyrolactone, propylene glycol monomethyl ether acetate, toluidine blue, Corning
glass slide (75 mm × 50 mm × 1 mm), triarylsulfonium hexafluoroantimonate salts (mixed,
50 wt. % in propylene carbonate), glass beads (size 75 μm, acid-washed) were obtained
from Sigma-Aldrich (St. Louis, MO). 1,1′-Dioctadecyl-3,3,′,′-tetramethylindocarbocyanine
perchlorate (DiI), Oregon Green 488 carboxylic acid diacetate, dithiothreitol (DTT),
ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0), Ca2+/Mg2+-free Hanks’ Balanced
Salt Solution (HBSS) were obtained from Invitrogen (Carlsbad, CA). EPON epoxy resin
1002F (fusion solids) was purchased from Miller Stephenson Chemical Co. (Sylmar, CA).
Biologic buffers and other regents were obtained from Fisher Scientific (Fairlawn, NJ).
Formulation of 1002F photoresist
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1002F photoresist was formulated by mixing 1002F resin, photoinitiator (triarylsulfonium
hexafluoroantimonate salts), and solvent (gamma-butyrolactone) as previously described.19
The weight ratio of resin:photoinitiator:solvent was 49:4.9:46.1 for formulation 10 (which
generates 10 μm thickness of film at the spin speed of 2000 rpm), 61:6.1:32.9 for
formulation 50, and 65:6.5:28.5 for formulation 100. The components were placed in a 500
mL brown glass bottle and the mixture was mixed by slowly rotating the bottle on a roller
system (Wheaton Science Products, Millville, NJ) until the resin was fully solubilized.
Colon crypt isolation and dye staining
Normal colon tissue, obtained from mice (129Sv/C57BL/6 strain), was rinsed 5 times with
20 mL HBSS. The tissue was then incubated in 1mM EDTA and 0.5 mM DTT in HBSS for
60–90 min to dissolve the surface mucosa. The tissue was rinsed 3 times with 20 mL PBS in
a 50 mL conical tube. 30 mL PBS was placed in the tube and it was then shaken vigorously
for 5 seconds. The detached crypts were then collected for immediate use, or fixed in 3.7%
formaldehyde in PBS and stored at 4 °C.
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To image the crypts by epifluorescence microscopy, the crypts were stained with DiI, an
orange-red fluorescent dye. A stock solution of DiI was made by dissolving DiI in 100%
ethanol at 0.5 mg/ml. For staining, 100 μL of the DiI stock solution was added to 500 μL of
colon crypt suspension in PBS (~50,000 crypts/mL), and the suspension was incubated at
ambient temperature for 60 min on a rotary shaker (Labquake, Barnstead/Thermolyne,
Dubuque, Iowa). After the incubation, the suspension was transferred to a centrifuge tube,
diluted with 10 mL PBS buffer, and centrifuged at 2000 rpm for 1 min. The supernate was
decanted and the pellet composed of the stained crypts was re-suspended in 10 mL PBS
buffer.
Fabrication of crypt surrogates
Figure 1A is a schematic of the fabrication process to create the crypt surrogates. A glass
slide was cleaned with deionized water and ethanol, and then dried with a stream of nitrogen
(Fig. 1A–i). A 1002F film of 150-μm thickness was obtained by spin-coating 1002F
photoresist (formulation 100) on the glass slide at 500 rpm for 10 s followed by 1500 rpm
for 30 s on a WS-200-4NPP spin coater (Laurell Technologies Corp.) (Fig. 1A–ii). The
coated slide was baked in a convection oven at 95 °C for 60 min. After cooling to room
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temperature, an SU-8 film of 10-μm thickness was spin-coated on the top of 1002F film
using SU-8 photoresist (formulation 10) at 500 rpm for 10 s followed by 2000 rpm for 30 s
(Fig. 1A–iii). The slide was baked in a 95 °C oven for 5 min. The film was then exposed to
UV light at a dose of 2000 mJ/cm2 through a photomask using an Oriel collimated UV
source equipped with a 350 nm short pass filter (PL-360-LP, Omega Optical, Brattleboro,
VT) (Fig. 1A–iv). The post-exposure baking was performed in a 95 °C oven for 16 min. The
sample was then developed in propylene glycol monomethyl ether acetate for 15 min, rinsed
with 2-propanol, and dried by a stream of nitrogen (Fig. 1A–v). To prevent the aggregation
of surrogates in water, the sample was treated with air plasma for 5 min using a plasma
cleaner (Harrick Plasma, Ithaca, NY). The structures were detached from the glass slide by
scraping using a single-edge razor blade, and then rinsed into a 50-mL centrifuge tube using
phosphate buffered saline (PBS). Since 10,000 surrogate structures were fabricated on a 20
mm × 20 mm area, a concentration of 400 surrogates/mL was obtained by suspending the
detached structures in 25 mL of PBS buffer.
Fabrication of an array of micromesh
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Figure 2A shows the schematic of the fabrication process for a micromesh array. A film of
1002F of 50-μm thickness was produced by spin-coating 1002F photoresist (formulation 50)
on a glass slide at 2000 rpm (Fig. 2A–ii). After baking at 95 °C for 40 min, the film was
exposed to UV light at a dose of 800 mJ/cm2 (Fig. 2A–iii). The post-exposure baking was
performed in a 95 °C oven for 10 min. The sample was then developed for 8 min, and baked
on a 120 °C hotplate for 60 min (Fig. 2A–iv). Finally, the freestanding film was released
from the glass slide by soaking the sample overnight in a soap solution (Fig. 2A–v). A 20 ×
20 array of capture sites was used for all experiments described.
Loading of colon crypts on the microdevice
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Figure 3 shows a schematic of loading crypts on the micromesh arrays. The freestanding
film was glued to a polypropylene tubing (ID = 15 mm, OD = 19 mm, length = 40 mm)
using PDMS prepolymer (10:1 mixture of base:curing-agent of Sylgard 184 kit) (Fig. 3A).
The PDMS was cured in a 75 °C oven for 60 min. The film was made hydrophilic by
treating it with an air plasma for 5 min. The tubing was then placed in a petri dish (diameter
× height = 55 mm × 14 mm). A specified number of crypts (see “Results”) were suspended
in 50 mL PBS buffer. The crypt suspension was added to the tubing and flowed by gravity
through the film where the crypts were captured by the capture sites in the micromesh array
(Fig. 3B). During loading, the liquid height was maintained at about 29 mm above the film
by adding PBS to the tubing. Excess liquid was removed from the petri dish during loading.
The microscope stage holding the device was gently tapped with a finger at ~ 1 Hz during
the loading procedure to aid in the alignment of the crypts during capture. After loading,
unfilled capture sites were plugged by settling glass beads in suspension (400 beads/mL)
onto the array (Fig. 3C).
Live crypts were loaded to the micromesh arrays immediately after the crypts were isolated
from colon (within 3 h of mouse sacrifice). To test the viability of the captured crypts, 1 mL
of Oregon green 488 carboxylic diacetate (20 μM) in PBS was added to the tubing (Fig. 3D).
After 10 min, the captured crypts were washed by 20 mL PBS to remove Oregon green
diacetate. The crypts were then examined on an inverted fluorescence microscope
(excitation/emission 470 nm/535 nm).
Measurement of dye movement across a crypt array
Toluidine blue (1% in PBS, 30 μL) was added to the luminal side (total volume 1 mL) of an
array. At varying times, 120 μL was removed out from basal side (total volume 19 mL). The
optical absorption (633 nm) of 100 μL of the liquid samples was measured using a
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SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). The concentration
of toluidine blue on the basal side (Cbasal) was then obtained from the optical density. The
concentration on the luminal side (Cluminal) was calculated from Cbasal, the total amount of
dye, and the liquid volumes of the luminal and basal reservoirs. Measurements were
performed on three separate samples to obtain an average toluidine blue concentration.
Optical micrographs
The samples were imaged under brightfield or standard epifluorescence microscopy using an
inverted microscope (Nikon TE2000-U). A fluorescein filter set (excitation/emission
470/535) was used for surrogate crypts and Oregon green diacetate stained crypts, while a
rhodamine filter set (excitation/emission 540/625) was used for the crypts stained with DiI.
Differential interference contrast (DIC) images were obtained using with a Leica SP2
microscope.
Scanning electron micrograph
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To image colon crypts using scanning electron microscopy (SEM), the suspension was
desalted as follows: 100 μL of fixed colon crypts (in PBS) was added to 10 mL distilled
water and centrifuged at 2000 rpm for 1 min. The supernate was decanted and the crypts
were then re-suspended in 10 mL distilled water. 100 μL of this suspension was then added
to a cover slip, and the sample was dried under ambient conditions. For the crypts captured
on the microdevices, salts were removed by a final rinse of DI water through the
microdevices. The various dried samples were coated with a 6-nm thick gold layer using a
sputter coater and imaged by SEM (FEI Quanta 200 ESEM, FEI Company).
Results and Discussion
Description of colon crypts and surrogates
Isolated colon crypts have a unique mushroom-shaped geometry. Generally the diameter of
the luminal (or apical) end is larger than that of the basal (or blind) end.20 Figure 1D shows
a micrograph of a typical colon crypt suspended in PBS, together with the debris of
epithelial cells or disrupted crypts. The isolated crypts displayed a wide size distribution
with a length of 241 ± 49 μm, a diameter at the luminal end of 100 ± 23 μm, and a diameter
at the basal end of 58 ± 10 μm (n = 20 crypts). Crypts stained with DiI were imaged under
fluorescence and DIC (Fig. 1D inset). The mushroom-shaped geometry of the crypt was
clearly demonstrated in the SEM image (Fig. 1E). It should be noted that shrinkage of the
imaged crypt occurred as a result of drying under the conditions used for SEM imaging.
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Due to the limited availability of isolated colon crypts, a surrogate model was created for the
initial testing of the microdevices. Figure 1A shows a simple process to fabricate the
surrogate crypts. Two layers of photoresists were sequentially spin coated on a glass slide.
The bottom layer (1002F of 150-μm thickness) was less photosensitive than the top layer
(SU-8 of 10-μm thickness). For photoresists of the same thickness, the UV dose generally
required for 1002F was 2–4 times higher than that for SU-8.19, 21 The two-layer film was
exposed to UV through a mask at a dose of 2000 mJ/cm2, which was 8-fold over-exposure
for the 10-μm SU-8 layer, and 2-fold over-exposure for the 150-μm 1002F layer. As a result
of the 4-fold greater exposure for the top layer of SU-8 compared to the 1002F layer in the
UV-exposed portion, the top layer was larger in diameter than the bottom layer resulting in a
mushroom-shaped structure (Fig. 1B and 1C) similar to the crypts. Although the opening on
the photomask was 40 μm, the UV overexposure caused the diameter of surrogates to be 91
± 6 μm for the top layer, and 54 ± 3 μm (n = 20 structures) for the bottom layer. The length
was 155 ± 7 μm. Compared with the isolated crypts the surrogates had greater uniformity
and geometry. The size of surrogates could be adjusted by fabrication conditions such as
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spin-coating speed, UV-exposure dose and photomask design. It is noted that the surrogates
were fluorescent when using a fluorescein-filter set because the SU-8 layer has 5–10 times
higher autofluorescence than the bottom 1002F layer (Fig. 1B inset).19 In addition,
surrogates to mimic the debris (ruptured crypts and clumps of dissociated cells) seen in
suspensions of the isolated crypts were created. The debris surrogates were composed of a
mixture of rectangular particles of 60 μm × 60 μm × 30 μm and 40 μm × 40 μm × 30 μm
(Fig. 1B) fabricated by exposing a 30-μm thick SU-8 photoresist to UV light through a mask
with 60 μm × 60 μm and 40 μm × 40 μm openings.
Fabrication of microstrainer and micromesh arrays
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To determine whether an array of colon crypts could be assembled, two different support
structures were fabricated, a microstrainer array and a micromesh. An array of
microstrainers was fabricated according to the process outlined in Fig. S1. Micromesh, a
simpler microdevice than the microstrainer, was fabricated according to the process outlined
in Fig. 2A. A freestanding film made from the commercial photoresist SU-8 was too brittle
to be used in the manipulation steps depicted in Fig. 3, which included film detachment from
a glass substrate as well as attachment of tubing to the final film. An alternative photoresist
1002F was more pliable than SU-8 and thus could withstand the manipulation steps of the
developed film.19 By soaking in a soap solution for 16 h, the film could be detached from
the glass as a freestanding structure. The detached film remained highly transparent. The
micromesh arrays were composed simply of circular holes. Arrays were created using a
variety of diameters (40–150 μm) for the openings. Figures 2B and 2C show SEM images of
a portion of a micromesh array composed of 80-μm capture sites. The freestanding
micromesh film made from 1002F was found to be resilient and did not fracture even after
bending and folding.
Loading colon crypts and surrogates onto the microstrainer and micromesh arrays
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The freestanding film composed of the microstrainer or micromesh array was glued to a
polypropylene tube that served as a fluid reservoir (Fig. 3A). The assembly was then treated
with an air plasma to make the surface of the film hydrophilic. This was necessary to
prevent air entrapment in the openings of the microstrainer or micromesh. Adding a
suspension of crypts or surrogates to the tubing reservoir resulted in fluid flow through the
openings in the film (Fig. 3B) that carried the suspended structures through the openings
where they were captured. The microstrainer was initially tested using crypt surrogates
mixed with debris surrogates. This structure captured the crypt surrogates, but capture sites
were frequently blocked by the debris surrogates (Fig. S2A), thus making individual sites
unavailable for capture. When the microstrainer was used to capture actual crypts, many of
the microstrainer capture sites were blocked by the epithelial debris present in the
suspension rather than captured crypts (Fig. S2B).
The simpler structure of the micromesh was then tested. The rationale for the micromesh
design was based on the geometry of the crypts. The smaller basal end of the crypt was
expected to enter the site under the influence of the fluid flow. In contrast, the larger luminal
end would prevent the crypt from entering the capture site luminal end first, and would also
act to retain a crypt that entered the site basal end first. The debris, which was generally
much smaller than the crypts, was expected to flow through the holes without being
retained. It was found in testing with surrogates (crypt and debris) that this was indeed the
case. Figures 4A and 4B show a portion of the micromesh array composed of 70-μm capture
sites before and after loading with surrogates. After loading with surrogates, 92 ± 3% of the
sites (n = 5 independent experiments) had captured surrogate crypts. No capture sites were
found to have been blocked by the surrogate debris. The surrogate loaded micromesh was
imaged by SEM on both faces (Fig. 4C and 4D). The SEM images demonstrated the capture
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of surrogate crypts by the capture sites with the luminal end hanging over the site on the
upper side of the film (Fig. 4C), while the basal end dangled out of the hole on the underside
of the film (Fig. 4D).
Since the micromesh was shown to effectively capture the surrogates, actual crypts were
then tested with this device. A suspension of fixed crypts was loaded on a device composed
of an array of 80-μm capture sites in the same manner as described above. Imaging by
brightfield and DIC suggested that the crypts were oriented vertically within the holes with
the basal end extending below the lower face of the array substrate (Fig. 4E, F). The
orientation of the crypts captured by the micromesh array was clearly shown by SEM
analysis of both faces of the film after crypt capture (Fig. 4G–J). Although the captured
crypts shrank due to vacuum drying of the sample for the SEM imaging, they remained in
their captured positions. The luminal end was shown to be trapped on the upper side of the
film, while the basal end extended out of the lower face of the film.
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Gentle tapping of the device with a finger during the loading process was found to be
important for properly orienting the crypts during capture. To be captured, the crypts must
be aligned with the fluid streamlines (Fig. S3A) such that the basal end flows into the
capture site ahead of the luminal end. When the crypts were settled on the micromesh in the
absence of vibration, only 12% ± 5% (n = 4 experiments) of capture sites were observed to
contain crypts in the proper orientation. Most of the crypts were oriented either horizontally
(crypts aligned perpendicular to the capture sites as shown in Fig. S3B or with the luminal
end in the capture site, but the basal end pointing upwards. In both of these orientations, the
crypt’s dimensions (150–250 μm length or ~100 μm luminal end) in comparison to the
smaller (~70 μm) mesh openings prevented the crypts from being properly captured in the
micromesh. It was found that vibration created by gently tapping the microscope stage that
held the device re-oriented un-captured crypts along the fluid flow with their basal ends
pointing toward the capture sites. In these studies, 94% ± 3% (n = 3 experiments) of
captured crypts were properly oriented. It should be noted that if the vibration was too
forceful, crypts that were already captured could be displaced from their capture sites, thus
reducing loading efficiency.
Loading efficiency of crypt surrogates and plugging of open capture sites
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To determine the loading efficiency, a suspension of crypt surrogates (400 surrogates in 50
mL PBS) was added to a micromesh array composed of 20 × 20 capture sites. The estimated
number of surrogates (400) used was equal to the number of capture sites making up the
array. In these studies, the loading efficiency was defined as the percentage of sites that
successfully captured the surrogates as verified by microscopic imaging. Gentle tapping as
described above was used to achieve the maximal loading efficiency. The loading efficiency
of crypt surrogates depended on the diameter of the capture sites, as illustrated in Figure 5A.
The optimal loading efficiency for surrogates was 92 ± 3% (n = 5) when an array of 70-μm
sites was used. Figure 5B shows an 8 × 10 portion of an array of 70-μm sites in which 76
sites captured crypt surrogates, and only 4 remained empty. A typical surrogate possessed a
diameter of 92-μm at the luminal end, and 53-μm at the basal end (Fig. 5A inset). The
loading efficiency was reduced when the diameter of the capture site was less than 70 μm or
greater than 90 μm. Not surprisingly, these data show that in order to capture the surrogate
the size of the site must be greater than the basal end and smaller than the luminal end of the
surrogate.
Although a high loading efficiency could be achieved for crypt surrogates, some sites
remained empty. Since the eventual goal will be to perform assays that expose captured
crypts to two different fluidic microenvironments on opposite sides of the array, the empty
sites must be plugged or sealed in order to prevent fluid exchange across the array. A simple
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way to plug these holes was found using physiologically inert glass beads of slightly larger
diameter than the capture sites in the array. A suspension of glass beads (100 beads in 1 mL
PBS, average diameter 75 μm) was added, and the beads loaded in the same manner as
crypts. Brightfield (Fig. 5D, E) and SEM images (Fig. 4C, D) showed the empty capture
sites firmly plugged by the glass beads. An excess number of glass beads were added to
ensure all capture sites that lacked crypts were plugged. The previously captured crypt
surrogates remained in place during the plugging procedure.
Loading efficiency of fixed crypts and plugging of open capture sites
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The loading efficiency of the isolated crypts was tested using a suspension of fixed crypts
(400 crypts in 50 mL PBS) added to a micromesh array in a manner identical to that
described in the previous section. The capture efficiency was 37 ± 5% (n = 5) for the crypts
when an array composed of 80-μm sites was used. It is likely that the greater size
distribution of the actual colon crypts as compared to the surrogates lowered the likelihood
of their capture compared to that of the surrogates. As with the surrogates, the loading
efficiency of the crypts depended on the diameter of the capture sites (Fig. 6A). Shown in
Figure 6B is a 6 × 8 portion of an array of 80-μm sites in which 28 sites held captured
crypts. A fluorescence image (Fig. 6C) of crypts stained with DiI clearly showed the
location of captured crypts. A typical crypt had a diameter of 113-μm at the luminal end, and
a diameter of 54-μm at the basal end (Fig. 6A, inset). A capture site diameter between 80 –
90 μm was found to provide the highest capture efficiency. Open sites could be plugged by
adding a suspension of glass beads (Fig. 6D, E) after the crypt capture procedure as
described in the previous section. Colon crypts have a relatively wide distribution in size, for
example, diameter of basal end is 58 ± 10 μm. This indicates that crypts of a certain size are
preferentially captured in the device. To capture crypts of different size, an array of holes
with a size distribution corresponding to that of the colon crypts could be utilized.
Formation of a gradient across the crypt array
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Formation of chemical gradients across the crypts is thought to be critical for proper colon
development and differentiation. For example, a gradient of gremlin (expressed by the
myofibroblasts underlying the intestinal epithelium) across the crypts is thought to be
necessary for maintaining Wnt signaling in the stem cell compartment at the base of every
crypt.22 To determine whether the crypt array could support a molecular gradient between
the luminal and basal crypt sides, toluidine blue was added to the luminal side of the array
(Fig 3D). Movement of toluidine blue was followed over time by measuring the absorbance
of the dye on the basal side (Fig S4). A molecular gradient of toluidine blue (Cluminal/Cbasal
>10) was formed that lasted over 8 hours, the longest time measured (Fig. 7A). Thus
chemical gradients can be formed across the crypt arrays and may be useful in the study of
crypt differentiation and polarization.
Capturing living colon crypts in the micromesh array
A long term goal for the current device is the formation of living crypt arrays for biomedical
experimentation. Since living crypts are softer and more flexible relative to fixed crypts, it
was important to assess whether they could be efficiently loaded into the arrays in a viable
state. To demonstrate whether living crypts could be arrayed in the micromesh array, freshly
isolated colon crypts were added to an array (400 capture sites, 80 μm holes). When 1,500
crypts were loaded onto the array, 80% of the capture sites were filled with crypts. Fig. 8A
shows 58 crypts captured in a 72-site segment. To determine whether the crypts remained
viable after placement on the array, Oregon green diacetate (a viability dye) was added to
the crypt array. All of the crypts loaded into the array actively metabolized the viability dye
to its fluorescent form (Oregon green) and concentrated the dye within the cellular
cytoplasm (Fig. 8B and C). This data suggests that an array of living crypts was formed.
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Conclusions
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Isolated colon crypts were captured in an oriented fashion on a simple device composed of
an array of micron-scale capture sites. It was found that a capture site composed of a simple
circular opening, termed a micromesh had several advantages for capturing the crypts over
an array composed of microstrainers. The micromesh was simple to create needing only a
one-step fabrication process. In actual use, the micromesh allowed debris to pass freely
through the capture site while the microstrainer frequently became clogged. To improve the
capture efficiency and orientation the isolated crypts were loaded onto the device as a
suspension and settled onto the array under the influence of fluid flow while a low frequency
vibration was applied. It is likely that the capture efficiency could be further improved by
sorting the crypts according to size before loading them on to the micromesh. The results in
the present study show the feasibility of using the micromesh to capture and form an array
of living colon crypts. A future goal is to load the crypts into an array within a microfluidic
device so that the luminal and basal sides can be accessed by distinct aqueous environments.
Such a device will facilitate in vitro studies of ion transport, stem cell differentiation and
other aspects of crypt physiology.
Acknowledgments
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We thank Robert Edwards (Department of Pathology, UCI) for providing the murine colons for experiments with
fixed crypts and for advice on crypt isolation and preparation. We also acknowledge Christopher Dekaney
(Department of Surgery, UNC) for providing murine colons for experiments with living crypts. This research was
supported by NIH (R01 EB007612 and R01 EB004436).
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Fig. 1.
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Surrogate crypts and colonic crypts (with debris). (A) Schematic of the fabrication process
for the crypt surrogate. (B) Brightfield and fluorescence (inset) images of crypt surrogate.
Debris surrogate is also shown. (C) SEM image of crypt surrogate. (D) DIC and brightfield/
fluorescence (inset) images of isolated colon crypts. Typical epithelial debris is also shown.
(E) SEM image of colon crypt. Scale bar is 50 μm.
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Fig. 2.
Micromesh array. (A) Schematic of the fabrication process for the micromesh. (B) and (C)
SEM images of the micromesh. The diameter of capture sites in the micromesh was 80 μm.
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Fig. 3.
Schematic of protocol for capturing colon crypts and for formation of a basal-luminal
chemical gradient. (A) A freestanding array of micromesh was glued to polypropylene
tubing. (B) A suspension of colon crypts was added so that the luminal side of the crypt was
contiguous with the lumen of the polypropylene tube. The crypt basal side was facing the
lower, larger reservoir. (C) A suspension of glass beads was added. (D) A gradient was
formed across the crypt array by adding a chemical to the luminal side.
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Fig. 4.
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Loading surrogates or colon crypts into the micromesh. (A–D) Loading of surrogates into a
micromesh array (diameter of opening = 70 μm). Brightfield images of a micromesh array
before (A) and after (B) loading with surrogates. SEM images of a loaded micromesh array
viewed from top (C) and bottom (D) sides. (E–J) Loading of isolated colon crypts on a
micromesh array (diameter of opening = 80 μm). (E) Brightfield image of a micromesh
array after loading. The crypts were found captured in the device oriented vertically and so
they appear dark due to blockage of light by the crypt tissue. (F) DIC image showing a
dangling crypt in a capture site viewed from the bottom face. (G–J) SEM images of a
micromesh array loaded with crypts viewed from top (G, I) and bottom (H, J). (I) and (J) are
close-up images of individual capture sites in “G” and “H”, respectively.
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Fig. 5.
Efficiency of loading crypt surrogates in the micromesh array. (A) Efficiency of loading
crypt surrogates vs. capture site diameter. The inset image shows the surrogate’s dimensions.
(B) Brightfield image showing the loading on an 8 × 10 portion of an array of 70-μm capture
sites. (C) Fluorescence image of “B” taking advantage of the autofluorescence of the
surrogates to clearly show the location of captured surrogates. (D–E) Brightfield images of
the upper (D) and lower (E) faces of the array with captured surrogates. Unfilled capture
sites (marked with an asterisk) were plugged with 75- μm glass beads.
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Fig. 6.
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Efficiency of loading isolated colon crypts in the micromesh array. (A) Efficiency of loading
crypts vs. capture site diameter. The inset image shows the dimensions of a typical crypt. (B)
Brightfield image showing a 6 × 8 portion of an array of 80-μm capture sites. (C)
Fluorescence image of “B”. (D–E) Brightfield images of the array with captured crypts
before (D) and after (E) unfilled capture sites were plugged with 75- μm glass beads.
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Fig. 7.
Formation of a toluidine blue gradient across the crypt array. Shown is the concentration of
toluidine blue on the luminal side (Cluminal) and basal side (Cbasal) vs. incubation time.
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Fig. 8.
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Loading live colon crypts on the micromesh. (A) Brightfield image of a micromesh array
after loading with live crypts (80 μm sites). (B) A close-up brightfield image showing a
dangling crypt in a capture site: basal end (*) and luminal end (#). (C) Fluorescence image
of the same crypt stained with Oregon Green 488 carboxylic acid diacetate. Scale bar is 50
μm for (B) and (C).
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