Author...........Signature redacted......................
Department of Materials Science and Engineering
April 24, 2015
Certified by..........Signature
red acted
Niels Holten-Andersen
Assistant Professor of Materials Science and Engineering
Professorship in Ocean Utilization
Dohe
Accepted by...................
Signature redacted
V..
Geoffrey Beach
Chair of the Undergraduate Committee
1
L)
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Fabrication of Tissue Scaffolds using Projection MicroStereolithography
by
Albert Keisuke Matsushita
Submitted to the Department of Materials Science and Engineering 7'
in partial fulfillment of the requirements for the degree of
J
Bachelor of Science in Materials Science and Engineering
at the
x
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
APRIL 2015 [une 20151-Massachusetts Institute of Technology 2015. All rights reserved.
cQ
2
Fabrication of Tissue Scaffolds using Projection MicroStereolithography
by
Albert Keisuke Matsushita
Submitted to the Department of Materials Science and Engineering
on April 24, 2015, in partial fulfillment of the requirements for the degree of
Bachelor of Science in Materials Science and Engineering
Abstract
In vitro liver models are a critical tool in pharmaceutical research, yet standard hepatocyte
cultures fail to capture the complexity of in vivo tissue behavior. One of the most critical features
of the in vivo liver is the extensive microvasculature which allows for the delivery of nutrients
and metabolites without exposing hepatocytes to de-differentiating fluidic shear stresses. A new
liver tissue scaffold design able to capture this histological organization may therefore improve
the functional longevity of seeded hepatocytes. The additive manufacturing technique of
projection micro-stereolithography (PuSL) proved capable of building non-cytotoxic and highly
complex 3D structures with microvasculature on the order of 20 um inner diameter. While
extensive biological testing remains to be carried out, the built structures reveal much promise in
PuSL as a method of tissue scaffold fabrication in terms of in vivo mimicking architecture.
Thesis Supervisor: Niels Holten-Andersen
Title: Assistant Professor of Materials Science and Engineering and Doherty Professorship in
Ocean Utilization
3
4
Acknowledgements
I would like to thank Micha S.B. Raredon and Professor Linda Griffith for their encouragement,
mentorship, and support, as well as the opportunity to work on this engaging and challenging
project. I would also like to thank all of the Course 3 faculty with whom I have had the pleasure
of learning, especially Professors Lorna Gibson, Linn W. Hobbs, and Niels Holten-Andersen. I
am also greatly indebted to John Rogosic, PhD, for his convincing me that getting a 49/100 on a
3.022 exam neither meant the end of the world nor that I literally understood less than half the
material in the class.
Lastly, I would like to thank my mother, sister, father, and Juan Carlos Ybarra for their love.
5
6
Contents
11
1 Introduction
1.1 Existing liver models...........................................................................11
1.2 In vivo liver architecture...................................................................
13
15
2 Technology Development
2.1 PuSL apparatus...............................................................................17
2.2 Point spread function ........................................................................
18
2.3 Resin delivery ...............................................................................
21
2.4 Resin development..........................................................................21
24
3 Protocol Development
3.1 Operation overview..........................................................................24
3.2 Vertical fabrication..........................................................................26
3.3 Stitch builds...................................................................................27
3.4 Multiple lens builds..........................................................................30
4 Discussion and Conclusion
32
References
33
7
8
List of Figures
1. The perfused multi-well array first described in 2010 by Domansky et al.
...... . . . . . . . . . 12
[7]
2. Native liver architecture. .......................................................................
13
3. An early, proposed CAD model mimicking native liver lobule architecture.............15
4. Schematic of projection micro-stereolithography, courtesy of the MIT Laboratory for
Manufacturing and Productivity
........................
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
16
5. Point spread function (PSF) quantifies the degree of "blurring" that occurs as a single
point of light is transferred through an optical system ...............
............. ....... . .
19
6. Additive exposures as a result of Gaussian light intensity distribution caused by PSF also
decrease negative feature resolution'].......................
. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 20
7. Substrate chamber with filter (left) and schematic...........................................21
8. Fig. 8 Divinyl Adipate (DAP) [A] and trimethylolpropane ethoxylate triacrylate (TMPE)
[B].................................................................................
.....23
9. Early constructs fabricated in PuSL using 35% DAP, 1 5% TMPE, 50% PEG200.........26
10. Achieving hexagonal close-packing of the scaffolds......................................
11. Early attempts to "stitch" hexagonal scaffolds...............................................29
12. Revised hexagonal scaffolds for stitched builds.............................................30
13. The addition of a base-layer (in blue) beneath the hexagonal scaffolds is proposed to
uncouple scaffold durability with feature resolution........................................31
9
28
List of Tables
1. Operating parameters of the Griffith lab PuSL as assembled by M.S.B.
Raredon.........................................................................................
2. Fabrication parameters for PuSL............................................................25
10
18
Chapter 1
Introduction
In vitro liver models are a critical tool in assessing drug metabolism and toxicity,
modeling disease processes, and investigating other areas of pharmaceutical research; yet,
standard hepatocyte culture conditions fail to capture the complexity of in vivo tissue behavior.
For instance, incorrect micro-environmental cues typical of a monolayer hepatocyte culture
(absence of perfusion flow, unmet oxygen demand, improper local stiffness, etc.) result in the
rapid loss of hepatocyte-specific functions including albumin secretion and drug metabolism.
Static cultures which drive hepatocyte aggregation into spheres or extracellular matrix (ECM)
gels prolong cellular function, but compromise mass transfer of nutrients, substrates, and
metabolites. An in vitro model that captures the liver's structural and functional complexity to
prolong hepatocyte viability and function is desired.
[12]
1.1 Existing liver models
To address the demand for an accurate in vitro liver model the Linda Griffith Lab has
previously developed and commercialized the LiverChipTM: a multiwell perfusion reactor to
support 3D liver cultures. The device comprises a polysulfone micro-machined main body with
12 wells, each of which accommodates a single 250um thick polystyrene (PS) scaffold with
300um diameter channels sitting atop a micron-porous filter (1-5um holes). Perfusion flow is
delivered downwards through independent circulatory loops by programmable miniature pumps
housed in the lower acrylic portion of the reactor. The filters provide resistance and uniformly
distribute flow. The filters also capture cells during initial seeding after which cells migrate to
the scaffold channel walls and assemble into tissue-like structures.[ 31
11
Fig. 1: The perfused multi-well array first described in 2010 by Domansky et al. Each of the
12 wells are fluidically isolated and under constant perfusion as delivered by programmable
miniature pumps housed in the lower acrylic portion of the reactor [b]. Polystyrene (PS)
scaffolds housed within each well (zoomed portion of [a]) provide the 3D physical support for
cell adhesion and tissue growth. Cells remained functionally viable for seven days as assessed by
immunostaining for hepatocyte phenotypic markers in the study 31
Rigid surfaces such as PS can drive 3D morphogenesis of liver cells: the seeded
hepatocytes in Domansky's work remained ftnctionally viable for a week on the collagen coated
PS scaffolds as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell
(LSEC) phenotypic markers.1 Substrate mechanical properties and nutrient permeability
influence hepatocyte function, however, and can potentially improve bioreactor performance. In
addition, PS is adhesive towards hepatocytes and as a result poor seeding efficiency is observed
as a large fraction of cells adhere to the top surface of the scaffold and fail to enter the channels.
Furthermore, the PS does not provide any liver-specific chemical cues to the cells that may aid
tissue formation. A more pliant scaffold that facilitates oxygen, nutrient, and waste diffusion that
can also be biochemically and structurally tailored is highly desirable. [4, 5,6
12
j
-
-
-
-
______________________
1.2 In vivo liver architecture
The liver cell's high metabolic activity necessitates perfusion to deliver oxygen and
nutrients in the existing scaffold design. The resulting fluidic shear stress, however, results in a
loss of cell morphology over time in the current bioreactor design.1 3 In contrast, in vivo liver
cells experience very little fluidic shear stress due to the architecture of the native liver
environment depicted below:
Centra
Hepakc
Ibue
-Portal lobule
.c
vein (C)
e
C
C
Porta
Sinusokdal
triad (P)
capillary
UVer
~canali
)
Hepatic a",ery
(brandi)
Portal vein
(branh)
Bile duct
Fig. 2: Native liver architecture. Each hexagonal prism 600-800 um across constitutes a liver
lobule, the functional unit of the liver. At the corner of each hexagonal prism lies two incoming
blood flows from the gut (the portal vein in blue) and the circulatory system (the hepatic artery in
red). The blood flows meet in the sinusoidal capillaries which empty into the central vein. [71
13
Each hexagonal prism comprises several liver lobules, the functional unit of the liver. At the
corners of each of the 600-800 um diameter hexagonal prisms lie two incoming blood flows
from the gut (the portal vein in blue) and the circulatory system (the hepatic artery in red). They
meet in the sinusoidal capillaries which empty into the central vein. Openings all along the
epithelial vasculature allow the fluid to enter interstitial spaces and bathe the cuboid hepatocytes.
This unique architecture and organization of cell types allows for the delivery of nutrients and
metabolites without exposing hepatocytes to de-differentiating fluidic shear stresses. A new liver
tissue scaffold design able to capture this histological organization may therefore improve the
functional longevity of seeded hepatocytes.
14
Chapter 2
Technology development
A more histologically faithful and pliant scaffold that facilitates oxygen, nutrient, and
waste diffusion is highly desired. To address this demand, graduate student Micha S.B. Raredon
of the MIT Linda Griffith lab proposed CAD structures mimicking native liver organization:
Fig. 3: An early, proposed CAD model mimicking native liver lobule architecture.
Biological engineers typically use etching, casting, and stamping techniques to fabricate microstructures. The initial scaffold design's three dimensional complexity (i.e. overhanging structures
in all three axes), however, necessitated free-form fabrication. Feature size on the order of
microns precluded material deposition 3D printing techniques which are typically limited at
positive feature resolutions of 50-300 um.
Under Dr. Nicholas Fang the MIT Laboratory for Manufacturing and Productivity has
developed projection-micro-stereolithography (PuSL): a free-form fabrication technique capable
of localized material patterning in less than 1-5 urn3 increments. Patterned UV light is
15
simultaneously projected onto photopolymer resin in layers to incrementally build 3D structures
as shown in the following schematic (courtesy of the Fang group):
A
UV beam
Beam
delivery
Z-axis stage
Digital micro-mirror display
Projection lens
CAD filej
UV photopolymer resin
8
ojection lens
Quartz window
Photopolymer resin
Resin chamber
Substrate
Fig. 4: Schematic of projection micro-stereolithography, courtesy of the MIT Laboratory
for Manufacturing and Productivity. A CAD file is sliced into micron-thick increments along
its z-axis. Each "slice" is converted to a bitmap image and delivered to the dynamic mask, or
digital micro-mirror display (DMD). UV light shined onto the DMD is reflected with the
appropriate pattern and reaches the photopolymer resin through a series of optics. [8]
16
A CAD file is sliced into micron-thick increments along its z-axis. Each "slice" is converted to a
bitmap image and delivered to the dynamic mask, or digital micro-mirror display (DMD). UV
light shined onto the DMD is reflected with the appropriate pattern and reaches the
photopolymer resin through a series of optics. Based on previous structures fabricated by the
Laboratory for Manufacturing and Productivity and discussion between Dr. Nicholas Fang and
Micha S.B. Raredon, it was concluded that PuSL would be able to fabricate structures similar to
the initial scaffold design (Fig. 3).
To work with PuSL, however, a specific photopolymer resin was required. The solution
had to be fluid and non-viscous at room temperature, and also needed to cure locally and quickly
for micron-scale features. The tissue-engineering application also meant the solution needed to
be biologically compatible before and after curing and allow the diffusion of glucose, oxygen,
cytokines, and large molecules such as serum albumin (-400 nm diameter).
2.1 PuSL apparatus
The PuSL apparatus was assembled my Micha S.B. Raredon under the guidance of
Professor Nicholas Fang to fulfill specific functional parameters: 1 ,'2 ]
17
Table 1: Operating parameters of the Griffith lab PuSL as assembled by M.S.B. Raredon
Resolution
(XY)
<2um
Resolution (Z)
< 0.5 um
Max sample
size
15 x 1 x 10 mm
Max speed
4 mm3 / hour (limited by resin viscosity)
UV light
365 nm, high intensity
Material
handling
PEG, PLGA, PCL, PLA, EG, initiators, acrylates, methacrylates,
common solvents
To allow fabrication on multiple length scales, two sets of projection lenses were installed: a
non-UV optimized lens with a 1:1 projection ratio (Canon) and one with a 10:1 reduction ratio
specifically tailored for UV light transmission (Zeiss). The UV light source was acquired from
Hamamatsu with peak output at 365 nm. The DMD chip was purchased from Texas Instruments
and of 1920x1080 resolution, 5 um pixel size. Additional components (beam splitter, camera,
beam expander, collimator, UV-coated mirror, and optical positioning equipment) were
purchased from Thorlabs and Edmund Optics. An Aerotech three linear encoder crossed-rolled
bearing stage with 50 nm step size was purchased for high repeatability and "stitch-and-repeat
builds" of multiple scaffolds on a single substrate.
2.2 Point spread function
The point spread function (PSF) refers to an optical system's response to an input of a
single point of light. It quantifies the degree of "blurring" that occurs such that an input step-
18
function of light intensity is spread into a Gaussian distribution of light intensity. The implication
for PuSL is that because the light intensity distributions are blurred, edges of smaller features
may fall below the cure threshold and fail to form. Wider features, on the other hand, generate
distributions of greater average intensity. They cure structures consistently albeit with a greater
risk of over-curing and the lateral spread of structures.
A
C
...
U.
I
.1
.
...
..
......
+
x
13
I
Fig. 5: Point spread function (PSF) quantifies the degree of "blurring" that occurs as a
single point of light is transferred through an optical system. The extent to which the stepfunction of intensity is spread into a Gaussian distribution indicates the quality of the optical
system.
19
Mr-EPOW
-_7i
. -
-.
- -
-
.-
____
-Jft_49
PSF further complicates the translation of CAD designs to cured structures when considering the
additive light intensity of spatially separate features: the Gaussian distributions overlap and
create non-zero light intensities in originally empty spaces. A resin with a low cure threshold
therefore loses negative feature resolution, while a resin with too high a cure threshold loses
positive feature resolution. The design of the scaffold structure and resin reactivity are
inextricably tied in optimizing a fabrication protocol.193
input
Image at print plane
Output
Fig. 6 Additive exposures as a result of Gaussian light intensity distribution caused by PSF
also decrease negative feature resolution. It is demonstrated with the example of tube
occlusion, however, that by fine-tuning threshold levels (tl, t2, and t3) different structures may
be fabricated with the same light intensity distributions. This may be done through the
manipulation of dye content as a photoabsorber.1"
20
2.3 Resin delivery
The printing chamber is sealed by a PDMS window bonded to quartz. Resin is delivered
through a channel in the PDMS accommodating 0.006" ID 316 stainless steel needle tubing
\
UV light
,
connected to a syringe pump.
Ouartz Qlass
Fig. 7: Substrate chamber with filter (left) and schematic. Courtesy of M.S.B. Raredon.
2.4 Resin development
The biocompatible resin should be suitable for high resolution PuSL fabrication and
allow fast difffusion of large biomolecules, such as albumin, once cured. The first criterion
requires the resin be a low viscosity fluid at room temperature which cures locally and quickly
when exposed to UV light. This would allow the fine, lobule-mimicking architecture that
inspired the project in the first place. The second criterion requires that the cured solid form
pores on the order of 100 nm diameter. This would allow the diffusion of large biomolecules out
of the sinusoid-inspired structures (Fig. 3) to deliver nutrients and metabolites to the hepatocytes
without exposure to de-differentiating fluid shear.
21
Poly(lactic-co-glycolic acid) (PLGA) was the initial resin candidate: in addition to its
biocompatible properties it is degradable, allowing for the scaffold to potentially be replaced by
natural tissue growth as it hydrolytically degrades. PLGA functionalized with dimethacrylate
was obtained from CM-Tek to allow for UV-activated cross-linking. Even at the lowest
molecular weight obtainable from the supplier (1840) the resin was too viscous.
Dimethylformamide, N-methyl-2-pyrrolidone, and tetrahydrofuran solvate dimethacrylate-PLGA
but are highly toxic.
In light of the problem of viscosity, a monomeric solution was adopted rather than a
polymeric one. Divinyl adipate (DAP) was selected as a candidate based on its use as a
crosslinker in biodegradable chitosan hydrogels although no previous study had demonstrated
the biodegradability of polymer networks made entirely of DAP (Dai, S. Xue. EnzymeCataylzed Polycondensation of polyester macrodiols with divinyl adipate). Porous solids were
cured from initial resin solutions containing a 50% DAP, 50% PEG 200 mixture, but the required
UV exposure was deemed to be too long. Trimethylolpropane ethoxylate triacrylate (TMPE, mw
428) was therefore added as a multi-arm acrylate molecule to accelerate the cross-linking process.
Iterations of various compositions were tested using a 10 um deep 10 point grid pattern until a
satisfactory resolution and cure time were achieved with a composition of 35% DAP, 15%
TMPE, and 50% PEG 200 with photoinitiator phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide
(Irgacure 819), a free radical generator. 1
22
H2
A
B
H2C
O
CH2
H
0
H2
HC
Fig. 8 Divinyl Adipate (DAP) [A] and trimethylolpropane ethoxylate triacrylate (TMPE)
[B]. The first successful iteration of the resin contained divinyl adipate (DAP) [A],
trimethylolpropane ethoxylate triacrylate (TMPE) [B], and polythylene glycol (PEG) in ethanol
solvent. [10, 11]
Due to Irgacure 819's lack of approval for biological use cytotoxicity tests were carried out on
scaffolds soaked in ethanol for 48 hours. Scaffolds were fabricated as those discussed in section
3.2. Seeding with iPS endothelial cells followed by three days of culturing resulted in nominal
degrees of cell death and excellent adhesion.
The addition of dyes further enhanced PuSL resolution: the presence of photo-absorbing
agents moderates the reactivity of DAP which otherwise would cure up to 50 um deep when
exposed under a 10 pixel wide line for 1 second. Several dyes were experimented with before
settling on Sudan I (absorption peaks of 418 and 476 nm) and Sudan 7B (absorption peak 364
nm) for their combined UV damping effect.1"
23
Chapter 3
Protocol Development
The PuSL itself was custom-built by Micha S.B. Raredon and as such had no built-in
fabrication software. The development of protocols for the fabrication of complex 3D structures
therefore began with simple confirmations of theory such as vertical fabrication and lateral
stitching before proceeding to more sophisticated builds as seen in Fig. 12.
The interaction of resin reactivity and scaffold design in PuSL fabrication necessitates
constant protocol re-evaluation for each unique combination of resin and scaffold: a scaffold is
designed in CAD, fabricated, and analyzed using stereo light microscopy, optical transmission
microscopy, and scanning electron microscopy (SEM). Stereo light microscopy allows
immediate inspection of fabricated structures in ethanol without dehydration. Transmission
microscopy reveals the extent of local curing as a function of material density. SEM is
ostensibly the most revealing technique, but limited in that it requires the drying of the samples
which distorts, buckles, and even fractures features.
3.1 Operation overview
Before fabrication, a 3D model in SolidWorks
TM
is exported as an STL file with
calibrated resolution. A 3D slicing program (Netfabb) divides the structure into cross-sectional
BMP files at regular intervals along the Z axis which form the images projected through PuSL.
Physical preparation of the fabrication process begins with the selection of a substrate.
PVDF filters would be typical of a scaffold designated for cell seeding, but glass coverslips may
be used instead to facilitate structure inspection in optical transmission microscopy. The
24
substrate is placed on the piston, or the floor of the fabrication chamber, and brought up within
20 urn of the print plane before being sealed off with the PDMS and quartz window. Resin is
delivered by syringe pump through the PDMS channel so as to either soak the filter or coat the
entire surface of the glass. The appropriate lens is secured in place, and fabrication may begin.
The variable parameters of the PuSL fabrication process are numerous, as summarized below:
Table 2 Fabrication parameters for PuSL
Experiment
Component
Parameter
Effect
Light source
Exposure
time
Increases degree, speed, depth, and lateral
spread of cure
Power
Increases degree speed, depth, and lateral
spread of cure
Focus
Cure uniformity
Lens
alignment
Feature alignment for structures fabricated
under different lenses
Stage
Bitmap image
X Y resolution Stitching of expusures
X Y stage
speed
Creates "streaked" features if improperly
adjusted with exposure program
Initial Z
height
Too high: no cure, substrate suction to
PDMS. Too low: structure fails to adhere to
substrate
Z step size
Increases lateral spread of cure
Z speed
Increases layer delamination
White/black
modulation
Light intensity (white level) and feature
definition (contrast)
Featurewidth Increases cure depth
Feature
Increases cure depth and lateral cure spread
arrangement
Material
Dye content
Decreases cure depth
25
-
3.2 Vertical fabrication
The first fabrication protocols were designed to build multiple, physically separate 3D
structures of unvarying cross-sections. The operating LabView code for these early builds shifts
the stage much like a typewriter: it exposes several points on the substrate along the X axis,
returns to the beginning of the row, moves down the appropriate row width, repeats the process
for Y rows, and finally lowers the Z stage to fabricate the next layer.
Fig. 9: Early constructs fabricated in PuSL using 35% DAP, 15% TMPE, 50% PEG200.
Images courtesy of M.S.B. Raredon.
These structures were fabricated using a resin composition without any dye to fine-tune curing
parameters (35% DAP, 1 5% TMPE, 50% PEG200). The hollow cylinders are 50 um in internal
diameter and are far too large to be accurate representations of the sinusoid microvasculature
depicted in Fig. 2. Nevertheless, the results were useful as early tests of the basic fabrication
program and initial resin.
26
-
"I
3.3 Stitch builds
Examining Fig. 9b one may notice that the scaffolds are printed as physically separate
units. Though useful to verify an initial protocol, such an architecture is unsuitable for seeding
hepatocytes due to the large amount of flat surface area onto which cells may adhere. These
areas would directly expose cells to large amounts of fluid shear and prevent access to the
microvasculature, resulting in significant attrition. It is necessary, therefore, to replicate the
hexagonally close packed liver lobule architecture (Fig. 2) through a "stitching" fabrication
protocol to protect cells from fluid shear and maintain them within diffusion distance.
New hexagonal scaffolds of 750 um inner circle diameter were designed to match the
600-800 um diameter of the liver lobule units. To achieve close-packing of the hexagonal
structures the initial LabView code was modified such that the stage would oscillate in the Y axis
during the printing of a single row:
27
X.1
Yzo
Fig. 10 Achieving hexagonal close-packing of the scaffolds. The LabView program was
altered to oscillate in the Y axis as it printed a row in the X axis. The exposure area moves from
point A-+ point B-* point C-- return to point A-+ point D.
The process results in a near-double exposure of UV light along the broad edges of the
hexagonal scaffolds. While this phenomenon allows for the overall scaffold to be held together
by well-cured and robust edges, it also diminishes the feature resolution along these lengths as
dictated by PSF (see section 2.2).
28
A
Fig. 11 Early attempts to "stitch" hexagonal scaffolds. The single unit (A) was designed such
that each corner provided a third of a complete sinusoid-mimicking tube when stitched together
in a hexagonal close-packed arrangement (B). The near doubled UV exposure along the edges
makes for well cured and robust structures (C) but also distorts the intended microvasculature
(D).
Based on the results of Fig. 11, fine features such as the sinusoid mimicking tubes were therefore
moved further inward to prevent tube occlusion and modified into whole tubes rather than thirds.
Accompanying this change in design was a change in the resin composition: dyes were added to
fine-tune curing as described in section 2.4. Multiple compositions were attempted before
arriving at one that successfully formed non-occluded tubes (Fig. 10 B, C): 35% DAP, 15%
TMPE, 50% PEG200, 2% Irgacure819, 0.05% Sudan I, 0.1% Sudan 7B.
29
A
Fig. 12 Revised hexagonal scaffold design for stitched builds. The initial hexagonal scaffold
design positioned thirds of cylinders meant to construct whole tubes when stitched together (Fig.
1 A). The doubled UV exposure at the edges caused by stitching, however, distorted these
features through additional curing (Fig. 1 D). The scaffolds were therefore redesigned to replace
the incomplete cylinders with whole tubes positioned further inward (A). Accompanying these
design changes were changes in the resin: dyes were added to fine-tune curing as described in
section 2.4. Multiple compositions were attempted before arriving at one that successfully
formed non-occluded tubes (B, C):
35% DAP, 15% TMPE, 50% PEG200, 2% Irgacure819, 0.05% Sudan I, 0.1% Sudan 7B.
3.4 Multiple lens builds
It was quickly identified that the preservation of feature resolution needed to be
uncoupled from scaffold robustness: greater UV exposure improved the durability of the
structures bonded along the edges of the hexagons but also caused tube occlusion. On the other
hand, early builds such as those shown in Fig. 1 OC ripped apart easily even in standing ethanol.
Therefore, it was proposed to create a base-layer onto which the main construct may be built.
One of the early such proposed designs is shown below:
30
Fig. 13 The addition of a base-layer (in blue) beneath the hexagonal scaffolds is proposed to
uncouple scaffold durability with feature resolution. Greater UV exposure improved the
durability of the structures bonded along the edges of the hexagons but also caused tube
occlusion. Early builds such as those shown in Fig. lOC ripped apart easily even in standing
ethanol. Therefore, it was proposed to create a base-layer onto which the main construct may be
built. This would require the use of both 1:1 and 10:1 lenses for a single build--a new challenge
in itself.'
The fabrication of such a base-layer using the 10:1 lens used for the scaffolds proper would take
an impractical amount of time, however. This limitation necessitated the use of both 1:1 and 10:1
lenses for a single build.
Switching lenses requires the manual swinging in and securing of each lens. It was found
that the lenses were misaligned but that a stage adjustment of -2.1 / +0.01 mm in the X and Y
axis, respectively, could compensate for that physical offset. The manual nature of the task,
however, also required high consistency in the manner of securing the lenses. To allow more
flexibility, the base-layer was re-designed to increase overlap between 10:1 and 1:1 exposure
areas.
31
Chapter 4
Discussion and Conclusion
Using projection micro-stereolithography, a resin, scaffold, and fabrication protocol were
developed in tandem for the construction of 3D hepatocyte scaffolds with features on
macroscopic and microscopic length scales. Unlike the traditional methods of etching, casting,
and stamping used by biological engineers to fabricate micro-structures, this new PuSL method
has demonstrated its ability to reliably fabricate overhanging structures in all three axes (Fig.
lOB). In addition, it provides far superior positive feature resolution of~15 um (Fig. 12C)
compared to other free-form fabrication technologies currently limited in the 50-300 um range.
While this method allows for the rapid prototyping of scaffold designs, the interactions
between the multitude of fabrication variables (Table 2) requires the careful iteration of protocols,
designs, and resins to arrive at successful builds suitable for tissue engineering. Much of the
groundwork for the final constructions of this thesis (Fig. 10, Fig. 11) is applicable to general
fabrication of other scaffolds (LabView codes, alignment calibrations, the introduction of a baselayer, etc). It is expected, however, that for each new specific tissue scaffold to be built on the
PuSL a new round of iterative perfecting will be required. That this can be done on the PuSL at
all, however, can also be considered one of the fabrication method's greatest strengths.
An aspect of optimization not fully explored in this work, however, is the aspect of
biological optimization. PuSL has proven to be able to fabricate non-cytotoxic and complex 3D
architectures, but as tissue scaffolds these structures must ultimately be evaluated through their
operational performance on cell seeding.
32
References
[1] Raredon, Micha S. B. Design and Fabricationof Physiologic Tissue Scaffolds Using
Projection-Micro-Stereolithography.Master's thesis. Massachusetts Institute of Technology,
2014.
[2] Shepard Neiman, Jaclyn A. et al. "Photopatterning of Hydrogel Scaffolds Coupled to Filter
Materials Using Stereolithography for Perfused 3D Culture of Hepatocytes." Biotechnology and
Bioengineering112. 4( 2015): 777-787.
[3] Domansky, Karel. et al. "Perfused Multiwall Plate for 3D Liver Tissue Engineering." Lab on
a Chip 10(2010): 51-58
[4] Coger, Robin. et al. "Hepatocyte Aggregation and Reorganization of EHS Matrix Gel."
Tissue Engineering3(1997): 375-390.
[5] Fasset, John. et al. "Type I Collagen Structure Regulates Cell Morphology and EGF
Signaling in Primary Rat Hepatocytes through cAMP-dependent Protein Kinase A." Molecular
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