SJSU-MastersWork

OPTIMIZATION AND QUALITY IMPROVEMENT OF BIOINKJET PRINTING
A Project Preparation
Presented to
The Faculty of the Department of General Engineering
San Jose State University
In Partial Fulfillment
Of the Requirements for the Degree
Master of Biomedical Devices Concentration
By
Ying-Chen Hsueh
Ganesh Iyer
Mirwais Sarwary
Dec 2011

Optimization and Quality Improvement of Bioinkjet Printing ii
© 2011
Ying-Chen Hsueh
Ganesh Iyer
Mirwais Sarwary
ALL RIGHTS RESERVED

Optimization and Quality Improvement of Bioinkjet Printing iii
SAN JOSE STATE UNIVERSITY
The Undersigned Project Committee Approves the Project Titled
By
Ying-Chen Hsueh
Ganesh Iyer
Mirwais Sarwary
APPROVED FOR THE DEPARTMENT OF GENERAL ENGINEERING
________________________________________________________________________
Dr. Maryam Mobed-Miremadi, General Engineering Department Date
________________________________________________________________________
Dr. Mallika Keralapura, Electrical Engineering Department Date
________________________________________________________________________
Dr. Leonard Wesley, MSE Director, General Engineering Date

Optimization and Quality Improvement of Bioinkjet Printing iv
ACKNOWLEDGEMENT
This project could not have been possible without Dr. Maryam Mobed Miremadi whom
not only served as our technical advisor but also served as a mentor and encouraged us
throughout this research. We would like to thank her for sharing her time, knowledge, resources,
and guidance. We would like to also thank Dr. Mallika Keralapura for her support as our
technical reader and Dr. Leonard Wesley for providing guidance and structure on how to present
ourselves and our project. Special thanks to Yiming Shan for her time and guidance towards this
project. Lastly, we would like to extend our thanks to the entire San Jose State University’s
faculty members of the Biomedical Device graduate program for all the support and opportunity
they have presented us.
Sincerely
Ying-Chen Hsueh
Ganesh Iyer
Mirwais Sarwary

Optimization and Quality Improvement of Bioinkjet Printing v
ABSTRACT
By
Ying-Chen Hsueh
Ganesh Iyer
Mirwais Sarwary
This study was undertaken to improve the overall quality and to determine the combination of
optimal parameters of the bioinkjet printing process for production of high quality miniaturized
alginate microcapsules. A parametric study using Taguchi L16 (45
) design of experiments was
used to achieve this goal. The 5 factors used are: Chitosan Concentration (%), CaCl2
concentration (%), jetting Frequency (Hz), Jetting Voltage (V), and the drop distance (mm).
Each of these factors was studied under a range of 4 predetermined levels. There were 50
replicates per run (20) that were leveraged towards the DOE analysis (overall 1000 readings).
The optimization types for each response were characterized by the following parameters:
Sphericity, Capsule Size, Turbidity, and Leakage or Strength. For each response, MINITAB
analysis yielded an ANOVA for the mean value and the Signal to Noise ratio (S/N) and the
recommended optimal levels were determined to be the ones with the highest S/N and closest to
the mean desired value. Therefore, the combined and optimal process parameters were
determined: CaCl2 (10% (w/v)), Chitosan (0.25% (w/v)), Frequency (600 Hz), Voltage (35V),
and Drop Distance (3mm). Using these parameters, the desired size, turbidity (% of
transmittance), and strength were achieved. Unfortunately, the confirmation run did not yield the
desired sphericity and it can contributed to the lack of print-head stability. Future work will
address this concern (using stable and clean print-head). A hypothetical business model based
on using the bioinkjet technology to address the diabetic needs is also discussed in this paper.

Optimization and Quality Improvement of Bioinkjet Printing vi
Table of Contents
ACKNOWLEDGEMENT............................................................................................................. iv
ABSTRACT.................................................................................................................................... v
List of Figures..............................................................................................................................viii
List of Tables .................................................................................................................................. x
1. INTRODUCTION .................................................................................................................. 1
2. MEDICAL APPLICATION................................................................................................... 2
2.1. ISSUE AND MARKET SHARE.................................................................................... 2
2.2. PHYSIOLOGY AND FUNCTION OF PANCREAS.................................................... 3
2.3. PYSIOLOGY AND FUNCTION OF KIDNEY............................................................. 4
2.4. STANDARD OF CARE................................................................................................. 6
3. BIOINKJET PRINTER .......................................................................................................... 6
3.1. WHAT IS A BIOINKJET PRINTER? ........................................................................... 6
3.2. BIOINKJET PRINTER CORE TECHNOLOGY .......................................................... 8
4. MICROCAPSULES ............................................................................................................... 9
4.1. APPLICATIONS OF BIOPRINTING ........................................................................... 9
4.2. WHAT IS MICROENCAPSULATION?..................................................................... 10
4.3. TYPES AND APPLICATION OF MICROCAPSULES ............................................. 10
5. PROPERTIES OF ALGINATE AND CHITOSAN............................................................. 12
6. DESIGN OF EXPERIMENTS ............................................................................................. 13
6.1. METHOD OF APPROACH......................................................................................... 13
6.2. CHEMICALS AND MATERIALS.............................................................................. 15
6.3. EQUIPMENT ............................................................................................................... 15
6.4. BIOINK PREPARATION............................................................................................ 16
6.5. RECEIVING SOLUTION PREPARATION ............................................................... 16
6.6. INKJET JETTING........................................................................................................ 16
6.7. MICROCAPSULE STRENGTH MEASUREMENT .................................................. 18
6.8. MICROCAPSULE VISCOCITY MEASUREMENT.................................................. 18
6.9. MICROCAPSULE pH MEASUREMENT .................................................................. 19
6.10. MICROCAPSULE MICROSCOPIC MEASUREMENT............................................ 20
7. RESULTS AND DISCUSSION........................................................................................... 21
7.1. DOE RESULT .............................................................................................................. 21
7.2. DOE ANALYSIS (NON-QUANTITATIVE) .............................................................. 22
7.3. DOE ANALYSIS (QUANTITATIVE)........................................................................ 24
7.3.1 STRATEGY................................................................................................................ 24
7.3.2. EFFECT OF PH AND ITS AFFECT ON TURBIDITY (%TRANSMITTANCE) ... 25
7.3.3. MICROCAPSULE SIZE ............................................................................................ 25
7.3.4. MICROCAPSULE SPHERICITY.............................................................................. 27
7.3.5. MICROCAPSULE STRENGTH................................................................................ 29
7.3.6. PERCENT TRANSMITTANCE OF RECEIVING SOLUTION .............................. 30
7.3.7. SYNOPSIS AND RECOMMENDED LEVELS........................................................ 31
8. CONFIRMATION OF COMBINED AND OPTIMIZED PROCESS PARAMETERS ..... 35
9. QUALITY IMPROVEMENT PROPOSALS....................................................................... 36
9.1. COVER DESIGN ......................................................................................................... 36
9.2. DISTANCE MEASUREMENT TECHNIQUE ........................................................... 38
10. ECONOMIC JUSTIFICATION....................................................................................... 39

Optimization and Quality Improvement of Bioinkjet Printing vii
10.1. EXECUTIVE SUMMARY .......................................................................................... 39
10.2. PROBLEM STATEMENT (DETAILED) ................................................................... 42
10.3. SOLUTION AND VALUE PROPOSITION (DETAILED)........................................ 43
10.4. MARKET SIZE (DETAILED)..................................................................................... 44
10.5. COMPETITORS AND MARKET GROWTH (DETAILED) ..................................... 44
10.6. BUSINESS MODEL (DETAILED)............................................................................. 46
10.7. COST (DETAILED)..................................................................................................... 47
10.7.1. PRODUCT 1 - DEVELOPMENT AND IMPLEMENTATION COST ............... 47
10.7.2. PRODUCT 1 - OPERATING EXPENSES & BREAKEVEN POINT................. 49
10.7.3. PRODUCT 2 - FDA AND CLINICAL TRAILS .................................................. 51
10.7.4. PRODUCT 2 - COST OF MICROENCAPSULATION....................................... 54
10.7.5. PRODUCT 2 - OPERATING EXPENSES & BREAKEVEN POINT................. 54
10.8. RETURN OF INVESTMENT (ROI) ........................................................................... 57
10.9. SWOT ANALYSIS ...................................................................................................... 57
10.10. EXIT STRATEGY........................................................................................................ 58
11. PROJECT DEVELOPMENT SCHEDULE ..................................................................... 60
12. CONCLUSION................................................................................................................. 61
13. FUTURE DIRECTIONS .................................................................................................. 61

Optimization and Quality Improvement of Bioinkjet Printing viii
List of Figures
Figure 1: Pancreas and Islet of Langerhans ................................................................................... 4
Figure 2: Kidney, Nephron, Glomerulus and filtration of blood ................................................... 5
Figure 3: Image of the inkjet printer. Arrows point to main components ...................................... 7
Figure 4: A thermal bubble inkjet print head and piezoelectric print head . .................................. 8
Figure 5: Microcapsules................................................................................................................ 10
Figure 6: Linear structure of alginate............................................................................................ 12
Figure 7: Calcium cation cross-linking of alginate...................................................................... 13
Figure 8: Structure of Chitosan..................................................................................................... 13
Figure 9: The inkjet printer setup and the control interface of the inkjet printer (MicroFab
Technologies, 2005)....................................................................................................... 17
Figure 10: The syringe/filter assembly & UV Spectroscopy Measurement ................................ 18
Figure 11: The type of viscometer used to measure bioink viscosity (IDES, 2010). ................... 19
Figure 12: Mettler Toledo pH measuring device ......................................................................... 20
Figure 13: Nikon Eclipse Ti Series Illuminator Microscope........................................................ 20
Figure 14: Size and Spherocity measurement techniques............................................................. 21
Figure 15: %Transmittance solution samples............................................................................... 22
Figure 16: CaCl2 (10%(w/v)) Chitosan (0%(w/v)) -400F-35-4mm ............................................. 23
Figure 17: CaCl2 (20%(w/v)) Chitosan (0%(w/v)) -600F-32V-3mm .......................................... 24
Figure 18: The plots of the main effects for means of microcapsule size. ................................... 26
Figure 19: Main effects plot for signal-to-noise (microcapsule size)........................................... 27
Figure 20: The plots of the main effects for means of microcapsule sphericity........................... 27
Figure 21: Main effects plot for signal-to-noise (sphericity)........................................................ 28
Figure 22: The plots of the main effects for means of microcapsule strength.............................. 29
Figure 23: Main effects plot for signal-to-noise (strength)........................................................... 29
Figure 24: The plots of the main effects for means of percent transmittance............................... 30
Figure 25: Main effects plot for signal-to-noise (percent transmittance). .................................... 31
Figure 26: DOE Analysis and Methodology for interpretation for % CaCl2 (w/v) ...................... 32
Figure 27: DOE Analysis and Methodology for interpretation for % Chitosan (w/v) ................. 32
Figure 28: DOE Analysis and Methodology for interpretation for Frequency (Hz) .................... 33
Figure 29: DOE Analysis and Methodology for interpretation for Voltage (V) .......................... 33
Figure 30: DOE Analysis and Methodology for interpretation for Distance (mm)...................... 34
Figure 31: Outcome of the DOE analysis- L16 Taguchi Method (MINITAB Analysis): Optimized
Process Window.......................................................................................................... 34
Figure 32: Microscopic analysis of capsules with blue dextran run with optimized parameter... 35
Figure 33: Microscopic analysis of capsules with optimized parameter. ..................................... 36
Figure 34: Cover Design for bioinkjet printer .............................................................................. 37
Figure 35: Latch and Hinge Specification .................................................................................... 37
Figure 36: Image of Cover Design with Bioinkjet printer............................................................ 38
Figure 37: Caliper device for measuring distance between the print-head tip and the receiving
solution........................................................................................................................ 39
Figure 38: Approach to Market..................................................................................................... 46
Figure 39: Revenue (Income) and expense chart for 2011 to 2021.............................................. 50
Figure 40: Net Profit/Loss Chart per Year.................................................................................... 50

Optimization and Quality Improvement of Bioinkjet Printing ix
Figure 41: Revenue (Income) and expense chart for 2011 to 2021.............................................. 56
Figure 42: Net Profit/Loss Chart per Year.................................................................................... 57
Figure 43: Product 1 and 2 product life cycle charts. ................................................................... 60
Figure 44: Research Project Schedule. ......................................................................................... 61
Figure 45: 5% CaCl2 (w/v) -No Chitosan (w/v) -300Hz-30V-1mm............................................. 82
Figure 46: 5% CaCl2(w/v) -0.25Chitosan (w/v) -400Hz-32V-2mm ............................................ 82
Figure 47: 5% CaCl2(w/v) -0.5Chitosan(w/v) -500Hz-35V-3mm ............................................... 83
Figure 48: 5% CaCl2(w/v) -0.75Chitosan(w/v) -600Hz-37V-4mm ............................................. 83
Figure 50: 10% CaCl2(w/v) -0.5Chitosa(w/v) -600Hz-30V-2mm ............................................... 84
Figure 51: 10% CaCl2(w/v) -0.75Chitosan (w/v) -500Hz-32V-1mm .......................................... 85
Figure 52: 15CaCl2(w/v) -0.75Chitosan(w/v) -400Hz-30V-3mm................................................ 85
Figure 53: 15% CaCl2(w/v) -0.25Chitosan(w/v) -600Hz-30V-1mm ........................................... 86
Figure 55: 15% CaCl2(w/v) -No Chotsan(w/v) -500Hz-37V-2mm ............................................. 87
Figure 56: 20% CaCl2(w/v) -0.25 Chitosan(w/v) -500Hz-30V-4mm .......................................... 87
Figure 57: 20% CaCl2(w/v) -0.5%Chitosan(w/v) -400Hz-37V-1mm.......................................... 88
Figure 58: 20%CaCl2(w/v) -0.75%Chitosan(w/v) -300Hz-35V-2mm......................................... 88

Optimization and Quality Improvement of Bioinkjet Printing x
List of Tables
Table 1: Number of people with diabetes (20-79 years), 2010 and 2030 ...................................... 2
Table 2: Types of microencapsulation techniques and its application. ........................................ 11
Table 3: List of factors and levels................................................................................................. 14
Table 4: Taguchi L16 fractional factorial experiment set-up......................................................... 14
Table 5: Jetting results of the L16 Taguchi matrix ........................................................................ 21
Table 6: Effect of pH and turbidity............................................................................................... 25
Table 7: Summary of the main effects for microcapsule size....................................................... 26
Table 8: Summary of the S/N ratios and the ranking of the main effects (microcapsule size). ... 27
Table 9: Summary of the main effects for microcapsule sphericity. ............................................ 28
Table 10: Summary of the S/N ratios and the ranking of the main effects (sphericity). .............. 28
Table 11: Summary of the main effects for microcapsule strength.............................................. 29
Table 12: Summary of the S/N ratios and the ranking of the main effects (strength). ................. 30
Table 13: Summary of the main effects for percent transmittance............................................... 30
Table 14: Summary of the S/N ratios and the ranking of the main effects (percent transmittance).
............................................................................................................................................... 31
Table 15: Costs of equipment ....................................................................................................... 47
Table 16: Cost of materials........................................................................................................... 48
Table 17: Labor cost ..................................................................................................................... 48
Table 18: Total cost breakdown and breakeven analysis.............................................................. 49
Table 19: FDA application fee ..................................................................................................... 53
Table 20: Cost of each batch of microencapsulation.................................................................... 54
Table 21: Total cost breakdown and break even analysis............................................................. 55

Optimization and Quality Improvement of Bioinkjet Printing 1
1. INTRODUCTION
Regenerative medicine is a new branch of medicine that attempts to change the course of
chronic disease and in many instances will regenerate or compensate for tired and failing organ
systems[1
].There is tremendous potential for the advancement of medical care through further
development of technologies that support regenerative medicine. One such technology is
cell/drug encapsulation via bioinkjet printing. Among other uses, cell encapsulation technology
is targeted to replace 27 percent of diabetes market, which is estimated (2010) to 101 billion
dollar market [2,3, 4, 5,6, 7
]. The primary equipment/technology used for cell/drug encapsulation is
the bioinkjet printer. Therefore the determination of optimal settings for the bioinkjet printer to
produce viable encapsulates is of the utmost importance and the core focus of this project.
Before the discussion of Design of Experiment (DOE) and the determination of the optimal
settings, the readers will be provided necessary background information with a better sense of
the justification for this project. The economic justification of bioinkjet printer is discussed with
respect to the medical need. Then the readers will be familiarized with the core technology
behind the bioinkjet printer, followed by the method of encapsulation. The paper also covers a
hypothetical business model of fabricating bioinkjet printer and its services and the eventual
release of a medical device that can potentially tap into the huge hybrid market of medical
devices and pharmaceuticals - addressing diabetes. The paper concludes with detailed
description of experiments, confirmation and a recommended set of optimal settings. This
process optimization is achieved by evaluating five critical parameters under four levels. The
data is evaluated under the Taguchi L16 method of Design of Experiments. The five critical
factors that have been identified are: the concentration of receiving solution: CaCl2 (%(w/v)) and
Chitosan (%(w/v)), Frequency (Hz) of jetting, Voltage (V) setting for jetting, and the Distance

(mm) from the print head to the receiving solution. The responses are the following:
Microcapsule Sphericity (Ratio), Size (µm), Percent transmittance of the receiving solution, and
the Strength of encapsulate. In addition, acidity (pH) and viscosity of the solution were
monitored to assess feasibility for future cell encapsulation work.
2. MEDICAL APPLICATION
2.1. ISSUE AND MARKET SHARE
Diabetes is the primary health issue that is being addressed in this paper. According to the
World Health Organization (WHO) it was estimated that last year (2010) 180 million people
were affected by diabetes, and projected a market of 360 million dollars for 2030 [8
]. The
breakdown of people affected by diabetes per country (2010 and 2030) is shown is the table 1.
Notice that India, China, and United States have the top three highest population of diabetes.
Table 1: Number of people with diabetes (20-79 years), 2010 and 2030 [9
]
The global health expenditure on diabetes was expected to total at least $376 billion in 2010
with an increase to $490 billion in 2030[10
]. Globally for 2010 it accounted for an astonishing
12% of the health expenditures [11
]. It is also estimated that diabetes accounts for an indirect

cost of $0.36 per dollar earned by the community. This indirect cost is due to lower productivity
and unemployment of people suffering from diabetes [12,13
]. The complications of diabetes are
many and serious. Just to name a few, diabetes are associated with serious complications such as
heart disease and stroke, hypertension, blindness, amputation, complications in pregnancy,
depression and kidney disease. With the two most common causes of kidney disease (Renal
disease) being diabetes and high blood pressure.
Kidney disease is currently ranked as the 9th leading cause of death in the US [14
].
Approximately 370k people in the US alone are suffering from kidney disease and the number is
expected to continue rising at 6%-10% annually [15
]. The annual cost of care for kidney disease
is approaching $100k per patient with a total cost exceeding $32 billion [16,17
].
Diabetes- type I (lack of insulin secreted by pancreas) and renal/kidney failures together
constituted to about 396 billion USD expenditure for 2010. Regenerative medicine using
encapsulation technique is targeted to replace 27 percent of this market which is estimated
(2010) to 101 billion dollar market [16,17
].
2.2.PHYSIOLOGY AND FUNCTION OF PANCREAS
The pancreas is a large gland that is located in the abdomen, posterior to the stomach, and
contains approximately one million clusters of cells known as the islets of Langerhans [18
].The
islets of Langerhans are a group of specialized endocrine tissue that make and secrete hormones.
Although it produces more than just insulin, the islets of Langerhans are commonly known as the
insulin-producing tissue [19
]. According to the Encyclopedia Britannica, the islet of Langerhans
is composed of five types of cells: Alpha cells, Beta Cells, Delta Cells, PPCells and D1 Cells.
As shown in the Figure 1, Alpha cells produce glucagon, which are used to raise the level
of glucose (sugar) in the blood. Beta cells are most numerous and are the source of insulin

production. The delta cells makes somatostatin which inhibits the release of numerous other
hormones in the body. The remaining two, PP cells and D1 cells, very little is known about their
function and role. Of these cells it is the degeneration of the insulin-producing beta cells that is
of most interest to us as this is the main cause of type I (insulin-dependent) diabetes mellitus. [12,
14
]
Figure 1: Pancreas and Islet of Langerhans [20, 21
]
2.3.PYSIOLOGY AND FUNCTION OF KIDNEY
As shown in figure 2, kidneys are bean shaped structures located near the middle of the back,
just below the rib cage, one on each side of the spine. It is composed of millions of nephorons
containing glomerulus [22
].

Figure 2: Kidney, Nephron, Glomerulus and filtration of blood [23
]
The primary responsibility of the kidney is the removal of waste through the process of blood
filtration. [15
] As figure 3 above shows, the removal of wastes occurs in tiny units inside the
kidneys called nephorons. As the cellular waste is carried into the kidney the blood is filtered at
the glomerulus, which contains tiny blood vessels (capillaries). The glomerulus allows extra
fluid and wastes to pass through while keeping normal proteins and cells in the bloodstream. A
complicated chemical exchange takes place, as waste materials and water leave the blood and
enter the urinary system. [15
] Most kidney diseases attack the nephorons, causing them to lose
their filtering capacity. The effect of the damage is usually not seen immediately but only after
years or even decades later. This failure is known as 'Renal disease' [13, 15
].

2.4.STANDARD OF CARE
What is the current standard of care for diabetes and renal disease? The largest market share
for treatment of diabetes & renal care is preventive care. Preventive care such as diet control,
exercise, and quitting smoking accounts for 73% of the market share [2,
3]. The second largest
market share, which accounts for 17.4%, is the medical device and monitoring industry – Insulin
pump, glucose monitoring and dialysis, etc. The remaining 9.6% of the market share belongs to
the pharmaceutical industry- drugs [4,
5]. The next technological breakthrough that will marry
the biomedical device to drugs is the technology of regenerative medicine. In regards to
regenerative medicine microencapsulation technique (via bioinkjet printer) seem to play a vital
and feasible role as the upcoming standard of care for diabetes and renal disease [6,24
].
3. BIOINKJET PRINTER
There are two essential features that make up a bio-inkjet printer—the bioink, a mixture of
living cells, treatment agents, and carrier gel [25
]; and the inkjet printer, a device used to generate
small droplets of bioink to form bio-membranes. This section will focus on the device and
highlight some of the essential features of an inkjet printer.
3.1. WHAT IS A BIOINKJET PRINTER?
A bioinkjet printer is essential a modified version of a commercial inkjet printer, which was
introduced in late 1980’s. What distinguishes an inkjet printer from other printers is that an
inkjet printer works by placing extremely small droplets of ink onto paper to create an image.
The dots that compose a particular print are usually between 50 and 60 µm in diameter [26
]. As a
reference the diameter of a human hair is approximately 70 µm. According to Jeff Tyson, inkjet
printers operate under two main categories of fundamental technology—non-impact technology

and impact technology. The basic difference is that an impact technology based printer must
have a mechanism that touches the paper in order to create an image whereas a non-impact does
not make physical contact.
Much of the early work, during the past ten years, has focused on the use of off-the-shelf
technology from commercial printers such as HP DeskJet [27
]. Therefore, an inkjet printer’s
ability to produce small droplets without the need of physical contact, and with high precision
make an inkjet printer ideal for applications such as computer-assisted deposition of
biomaterials.
The figure below is a photo of the bioinkjet printer used in our study, which consists of a
CCD camera (30 fps), a control unit, a piezoelectric print-head (60 µm aperture), a triggering
unit, a fluid delivery unit, an air jet unit, and a PC equipped with MicroFab JetServer software
package.
Figure 3: Image of the inkjet printer. Arrows point to main components
The software is used to control the waveform parameters, which consists of rise (µsec), dwell
time (µsec), fall (µsec), echo (µsec), final rise (µsec), frequency (Hz), voltage (V), and
drops/trigger. The droplet generation begins with the triggering box sending signal to the inkjet

control unit and CCD camera control PC. The solution is jetted through the print-head into the
receiving solution. The CCD camera captures the droplets as it is ejected out of the print-
head.[28
]
3.2.BIOINKJET PRINTER CORE TECHNOLOGY
Currently there are two main inkjet technology used to form the droplets of ink—a thermal
bubble inkjet print head and piezoelectric print head. Printers such as Cannon and HP use
thermal heads to produce droplets. The ink is repeatedly heated to very high degrees of
temperature for short bursts of time to generate a vapor bubble in the ink reservoir [29
]. As the
ink expands the droplet is ejected out of the nozzle (see Figure 4). Heat is the main drawback of
this method for use in bioinkjet printing. The vaporization of micrometer-sized layer of liquid in
contact with the thin film resistor may subject the cells to a 5 microsecond period of heat and
stress [30
]. There are several researchers that have contributed to the increasing evidence that
heat shock has long-term effects on electrophysiological properties of neurons [31
, 32
]. Although
other researchers, such as Xu et al., confirmed in their study that the heating process of thermal
bubble inkjet does not significantly alter the living cells membrane properties [33
] It is important
to note that manufactures, such as Ulano, do state that the cooling phase of this method alters the
critical nozzle alignment [34
].
Figure 4: A thermal bubble inkjet print head and piezoelectric print head [35
].

Alternative to the thermal bubble inkjet print head is the piezoelectric print head.
Piezoelectric print head does not use heat to generate droplets and therefore operates under
ambient temperature. Because the print head is not exposed to excess heat it is not subject to
nozzle misalignment or any other critical dimensional change (nozzle inner diameter) due to
heat. As shown in figure 2, above, in piezo driven devices, the drop size is controlled by the
waveform sent to the print head [36
]. A piezo technology uses precise electrical pulses to cause
the ink reservoir wall in the head to compress, thereby projecting ink out through the nozzle.
The applied voltage is pulsed, which causes a Rayleigh mode wave to propagate along the
surface. This causes the liquid, bioink, to also propagate along the surface, and the wave causes
some of the liquid to splash off the surface as droplets [37
] Programming can be used to control
the exact placement, size, and shape of each droplet [38
].
4. MICROCAPSULES
4.1.APPLICATIONS OF BIOPRINTING
Bioprinting can be classified into several categories, such as atomization and inkjet printing.
Atomization is a method, which cross-links the polyelectrolytes to form hydrogels. The purpose
of using inkjet printing method is to miniaturize the microcapsules and hence improve the
diffusion rate of compounds in and out of the capsule. Microcapsules of smaller diameters have
the potential of being more effective than the larger microcapsule, such as oral therapeutic or
drug delivery system [39
]. For diabetes, the currently available insulin delivery products are not
convenient. Therefore, alginate can be employed for encapsulating islet cells. Islet cells in these
microcapsules produce insulin and pores in the capsule’s membrane which can release insulin
constantly.

4.2.WHAT IS MICROENCAPSULATION?
Microencapsulation is a technique by which an inactive secondary material encloses an
active compound- solid, liquid or gas. The purpose of shielding the active compound is to
protect it from surrounding environment. The active material is called ‘Core’ where as the
enclosing secondary material is called ‘Shell’ as shown in Figure 5.
Figure 5: Microcapsules
The microencapsulation technique dates back to middle of twentieth century when Schleicher
and Green produced dye in a microencapsulated state, for the manufacture of carbonless copying
paper. [40
, 41
] With the evolution and wide spread usage of this technique microencapsulation
found many applications from chemical to cosmetic and now in pharmaceutical and biomedical
industry [42
].
4.3.TYPES AND APPLICATION OF MICROCAPSULES
Microcapsulation technique for use in the medical industry can provide advantages that other
methods may not be able to achieve. For the pharmaceutical or the biomedical industry,
encapsulation of bioactive materials such as drug compound or various cells (such as insulin
producing beta cells) can be encapsulated to provide the following unique advantages: An
encapsulated cell is protected from the host system by the inert membrane; an encapsulated cell

is invisible to the host system and therefore will not entice the host immune system; an
encapsulates semi-permeable membrane allows for a controlled release of the active compound
to its surrounding. Encapsulation techniques are divided into three major categories (as shown in
Table 2):
1. Chemical method: Starting materials are monomer or prepolymers. Encapsulation
process involves chemical reactions.
2. Ionotropic method: Starting material is a polyelectrolyte, polymers whose repeated units
have an electrolyte group, also known as polysalts.
3. Physical/Mechanical method: Starting material is a polymer where no chemical reactions
are involved and only shape fabrication takes place.
Table 2: Types of microencapsulation techniques and its application.
Microencapsulation Types Subtypes Applications
Chemical methods
Suspension Polymerization Textile
Emulsion Polymerization Drug delivery
Dispersion Bioscience
Interfacial Crop protection, Drug delivery
Ionotropic method
Ionotropic gelation or polyelectrolyte
complexation
Tissue Engineering, Regenerative
Medicine, Probiotics, Drug Delivery
Physical/Mechanical methods
Suspension cross linking Drug delivery
Solvent evaporation/extraction Drug delivery
Phase separation Drug delivery
Spray drying Food Technology
Fluidized bed coating Food Technology
Melt solidification Food Technology
Precipitation Biocatalysts
Co-extrusion Biomedical
Layer by layer deposition Biosensor
Supercritical fluid expansion Drug delivery
Spinning disk Food Engineering
Our research focuses on the Ionotropic chemistry, which is the standard method in
regenerative medicine [43
,44
]. Traditionally, atomization is used to jet hydrogels into a cross-
linking solution where ionotropic gelation occurs. More recently, inkjet printing has been used

to generate smaller droplets to form bio-membranes. The encapsulant material used for this
study are natural polymers like polysaccharides alginate because alginate is a non-toxic,
biodegradable, naturally occurring polysaccharide obtained from marine brown algae, certain
species of bacteria [45
].
5. PROPERTIES OF ALGINATE AND CHITOSAN
The bioink for bioprinting could be a heterogeneous mixture of buffered hydrogels, and other
compounds of interest. The linear structure of alginate is shown in Figure 6. Alginate is soluble
in water and capable of forming hydrogel in the presence of divalent cations, such as calcium
cations (Figure 7). Hydrogel are hydrophilic networks capable of mimicking biological tissues
and absorbing a large amount of biological fluid or water. Typically, alginate membrane is not
permeable. However, after being treated with sodium citrate, it becomes semi-permeable which
allows drugs or other small molecules to move in and out.
Figure 6: Linear structure of alginate.

Figure 7: Calcium cation cross-linking of alginate.[46
]
Chitosan is plentiful in the exoskeletons or hard shells of crustaceans. It is an unbranched
copolymer of D-glucosamine and N-acetyl-D-glucosamine (Figure 8) that is derived from chitin.
Alginate carries negative charges and can interact with positively charged chitosan, forming
physical cross-linked hydrogels complex. Alginate-chitosan complex reduces tendency of
swelling and improves structural strength and mechanical stability.
Figure 8: Structure of Chitosan.[47
]
6. DESIGN OF EXPERIMENTS
6.1.METHOD OF APPROACH
The Design of Experiments (DOE) technique enables us to determine the relationship between
parameters that affect the jetting process. It also helps us determine the important factors that

need to be controlled through the microcapsule formation process. An L16 designed comprised
of 5 factors, 4 levels was used [48
,49
, 61
].
Table 3: List of factors and levels.
Factors
Levels
Level 1 Level 2 Level 3 Level 4
Chitosan concentration (%) 0 0.25 0.5 0.75
CaCl2 concentration (%) 5 10 15 20
Frequency (Hz) 300 400 500 600
Voltage (V) 30.5 32 35 37
Distance (mm) 1 2 3 4
Through standard DOE an experiment to test all combinations of parameters (45
) would
require one thousand and twenty-four (1024) separate experiments. This would be prohibitively
long and expensive. To overcome this, a collapsed fractional factorial designed experiment
known as L16 Taguchi matrix (16 total runs) was performed. The parameter level combinations
are shown in Table 11.
Table 4: Taguchi L16 fractional factorial experiment set-up.
Runs
Standard
Order
CaCl2
(%)
Chitosan
(%)
Frequency
(Hz)
Voltage
(V)
Distance
(mm)
1 5 0 300 30.5 1
2 5 0.25 400 32 2
3 5 0.5 500 35 3
4 5 0.75 600 37 4
5 10 0 400 35 4
6 10 0.25 300 37 3
7 10 0.5 600 30.5 2
8 10 0.75 500 32 1
9 15 0 500 37 2
10 15 0.25 600 35 1
11 15 0.5 300 32 4
12 15 0.75 400 30.5 3
13 20 0 600 32 3
14 20 0.25 500 30.5 4
15 20 0.5 400 37 1
16 20 0.75 300 35 2

To determine the appropriate Taguchi matrix the degree of freedom (DOF) of the matrix was
calculated by the following formula [50
]:
DOF = 1+ [#factors *(#Levels-1)] = 1+[5*(4-1)] = 1+[5*3]=1+15 =16.
Thus Taguchi’s L16 (16 run DOE) was chosen. Taguchi method randomly picks the 16 runs
thereby providing the same level of process parameter details in less number of run (16 runs vs.
1024).
6.2.CHEMICALS AND MATERIALS
The following list of chemicals is needed for the production of microcapsules:
 Sodium Chloride (Fisher Scientific, AC19409)
 Calcium Chloride (Fisher Scientific, AC12335)
 Low Molecular Weight Sodium-Alginate (Sigma Aldrich, A0682)
 Chitosan (Sigma Aldrich, 448869)
 Blue Dextran (MW 2000000) (Sigma Aldrich, D4772)
 Deionized (DI) Water (San Jose State University)
 Nitrogen Gas (San Jose State University)
 Sodium Acetate (Baker Analyzed Reagent, 3460)
 Acetic Acid (Mallinckrodt U.S.P, 2504)
6.3.EQUIPMENT
In addition to the chemicals and materials the following lab equipment are needed to perform
the necessary experimentations:
 Bioinkjet Printer and accessories (Microfab Technologies)
 Cannon-Ubbelohde capillary viscometer (Capillary size 150)
 Contact Angle Measurement Device (SJSU design)

 Nikon Epiphot 40 Microscope
6.4.BIOINK PREPARATION
The bioink used for the jetting compartment was formulated using the following: 0.5% low
molecular weight sodium-alginate mixed with 0.4 mg/ml dextran blue. The bioink viscosity was
around 5 cp.
6.5. RECEIVING SOLUTION PREPARATION
The receiving solution was made of CaCl2 and Chitosan. Multiple concentrations of CaCl2
and Chitosan were tested in this project. For Chitosan preparation, 0.4g Chitosan was added
to10.2ml 0.1N acetic acid and 9.8ml 0.1N sodium acetate to make a 2% Chitosan solution. The
pH was adjusted to 4.6.
6.6.INKJET JETTING
Jetting was performed using the following steps:
1) The “MFJ Jet Server” program was started by double-clicking its icon in Disk C.
2) The system was rinsed with DI water and 0.9% NaCl (w/v) solution for three times.
3) For each wash solution the filter (Whatman®
disposable filter 1.0 µm) was changed.
4) The bioink was added to the syringe.
5) The vacuum was turned on.
6) The back pressure was adjusted to keep the fluid from dripping. The back-pressure is
around -25 psi.
7) The receiving solution was placed into a Petri dish, and the nitrogen gas flow was
started to stir the solution.

8) The height of the Petri dish was adjusted, and the tip of the print head was kept about
1-4 mm higher than the receiving solution.
9) Click on the Start jetting button in the control interface (Figure 9).
Figure 9: The inkjet printer setup and the control interface of the inkjet printer
(MicroFab Technologies, 2005).[51
]

6.7. MICROCAPSULE STRENGTH MEASUREMENT
The following steps were performed to measure the microcapsule strength.
1) Get a syringe and attach it to a one micron filter.
2) Place a mini-bottle under the syringe/filter (Whatman®
disposable filter 1.0 µm)
assembly.
3) Remove the piston and pour the contents of the receiving solution containing the
capsules in a syringe (Figure 10).
4) Let contents go through passively without pushing with the piston. Wait for thirty
minutes.
5) Test the solution for dextran blue using the Agilent 8453 UV-Visible Spectroscopy
System Measurement.The wavelength used for our application is 600 nm. The
version of the software being B.04.1.
Figure 10: The syringe/filter assembly & UV Spectroscopy Measurement [52
].
6.8.MICROCAPSULE VISCOCITY MEASUREMENT
The Ubbelohde capillary viscometer (Size 150), produced by Cannon Instrument Company,
is used to measure the viscosities of solutions, shown in Figure 11. The viscosity measures the

time that it takes a suspended solution to drop through a certain volume. The calculation gives
the kinematic and dynamic viscosity of a solution. It has been aimed to keep the formulation
viscosities under 5cP.
Figure 11: The type of viscometer used to measure bioink viscosity (IDES, 2010).
6.9.MICROCAPSULE pH MEASUREMENT
The measuring tip of the Mettler Toledo pH device was dipped into the receiving solution
(CaCl2 (%(w/v)+ Chitosan (%(w/v)) to get the readings. The Figure 12 as below shows the
device used for the measurement.

Figure 12: Mettler Toledo pH measuring device [53
]
6.10. MICROCAPSULE MICROSCOPIC MEASUREMENT
The following equipment- Nikon Eclipse Ti Series Illuminator Microscope with version
NIF 3.2.2 was used to capture the images of the capsules (both normal encapsulants and also
fluorescent encapsulants).
Figure 13: Nikon Eclipse Ti Series Illuminator Microscope.
The image as below shows the technique on how the Spheroicity and Size were measured.

Figure 14: Size and Spherocity measurement techniques.
7. RESULTS AND DISCUSSION
7.1.DOE RESULT
The jetting results of the L16 Taguchi matrix are shown in Table 5.
Table 5: Jetting results of the L16 Taguchi matrix

Some of the sample images as shown below reflect the level of %transmittance as captured. The
numbers with 90’s and above were transparent and solutions in 60’s (15% CaCl2 (w/v) + 0.25%
Chitosan (w/v)) and in single digit were translucent or very whitish in nature.
Figure 15: %Transmittance solution samples.
As shown in the tables above there were 4 reruns to match the response and the outcomes with
the previous runs. They being 10% CaCl2 (w/v), 0% Chitosan(w/v), 400 Hz Frequency, 35V
voltage and 4 mm distance; 10% CaCl2 (w/v), 0.35% Chitosan (w/v), 300 Hz Frequency, 37V
voltage and 3 mm distance; 10% CaCl2 (w/v), 0.5% Chitosan (w/v), 600 Hz Frequency, 32V
voltage and 2 mm distance & 20% CaCl2 (w/v), 0% Chitosan (w/v), 600 Hz Frequency, 32V
voltage and 3 mm distance. After the reruns-outcomes /responses were validated with the
previous runs-outcomes/responses.
7.2.DOE ANALYSIS (NON-QUANTITATIVE)
The following were the microscopic observations while performing the DOE experiment.
 Circular Shape of Encapsulants with respect to Receiving Solution
It was observed across the entire DOE that irrespective of frequency, distance of the
bioink jet needle to receiving solution , voltage and even the % of CaCl2 (w/v), the increase
of Chitosan concentration into CaCl2 for receiving solution was lowering the sphericity.

 Transparency of the receiving solution
It was observed that 0.25% Chitosan (w/v) (or less than 0.25% Chitosan (w/v)) with
15% CaCl2 (w/v) (or less than 15% CaCl2 (w/v) till 5% CaCl2 (w/v)) seems to be the limit
for the receiving solution to maintains its transparency. Beyond these limits the receiving
solution tends to be less transparent or more translucent in color and hence difficult to
detect the capsules.
 Capsule Formation (with respect to % Chitosan (w/v) in CaCl2 (w/v)):
Irrespective of the Frequency, Distance and Voltage, it was observed that 0.5%
Chitosan (w/v) (or less than 0.25% Chitosan (w/v)) with 15% CaCl2 (w/v) (or less than
15% CaCl2 (w/v) till 5% CaCl2 (w/v)-except 20% CaCl2 (w/v) with 0% Chitosan (w/v))
seems to be the limit for the formation of the Capsules.
 Images from each of the DOE Runs:
Sample images of capsules from the DOE runs are as mentioned below.
Figure 16: CaCl2 (10%(w/v)) Chitosan (0%(w/v)) -400F-35-4mm

Figure 17: CaCl2 (20%(w/v)) Chitosan (0%(w/v)) -600F-32V-3mm
7.3.DOE ANALYSIS (QUANTITATIVE)
7.3.1 STRATEGY
There were 50 replicates per run that were leveraged towards the DOE analysis (overall
1000 readings).The optimization types for each response were characterized by the following
parameters:
 Sphericity: Nominal the best. Closure to 1 the better.
 Capsule Size: Nominal the Best
 Turbidity: Smaller the better or % transmittance higher the better.
 Leakage: Smaller the better. This response was not analyzed because the detected
leaked amounts by spectrophotometry were within the 3SD of the control (blank
sample).
 For each response, MINITAB analysis yields an ANOVA for the mean value and the
Signal to Noise ratio (S/N). For the most important factors by response (as indicated
by the ANOVA ranking in Table 7-14) the recommended levels are the ones with the
highest (S/N) and closest to the mean desired value.

7.3.2. EFFECT OF PH AND ITS AFFECT ON TURBIDITY
(%TRANSMITTANCE)
From the table as shown below there wasn’t a directly co-relation observed between pH
and turbidity (%transmittance) but it was observed that %CaCl2 (w/v) had more influence on
turbidity (%transmittance) than % chitosan (w/v).
Table 6: Effect of pH and turbidity
%CaCl2
(w/v)
%Chitosan
(w/v)
% Transmittance pH
15 0.75 1.61 2.9
15 0.5 1.65 3.73
20 0.5 1.67 3.71
20 0.75 1.72 4.03
20 0.25 2.49 4
15 0.25 68.59 4.05
10 0.75 91.94 4.13
5 0.75 92.01 3.1
10 0.5 95.08 3.8
5 0.25 96.80 4.1
5 0.5 97.35 4.37
10 0.25 98.23 4
15 0 98.83 6.3
10 0 99.04 3.77
20 0 99.55 6.45
5 0 99.69 5.48
7.3.3. MICROCAPSULE SIZE
For the characteristic of microcapsule size, Figure 18 and Table 7 below display the model of
the main effects for means and the means ranking importance of the variables.

Figure 18: The plots of the main effects for means of microcapsule size.
Table 7: Summary of the main effects for microcapsule size.
Level CaCl2 (%) Chitosan (%) Frequency (Hz) Voltage (V) Distance (mm)
1 45.55 35.74 40.13 38.75 43.87
2 41.88 46.62 38.42 39.89 44.19
3 42.53 40.79 46.95 41 41.09
4 29.23 50.69 42.12 48.84 39.7
Delta 16.32 14.95 8.54 10.08 4.49
Rank 1 2 4 3 5
Based on the table and figure above, the variables % CaCl2 (w/v), %Chitosan (w/v) and
Voltage (V) had the greatest effect on the variation of microcapsule size. Also for the
microcapsule size characteristic, Figure 19 and Table 8 below display the summary S/N ratios
and the ranking of the main effects.

Figure 19: Main effects plot for signal-to-noise (microcapsule size).
Table 8: Summary of the S/N ratios and the ranking of the main effects (microcapsule size).
1 23.13 22.61 21.86 21.04 21.99
2 23.46 25.95 24.07 23.08 24.89
3 23.62 22.12 23.37 23.41 24.52
4 24.16 23.38 24.4 25.59 22.4
Delta 1.03 3.84 2.54 4.55 2.89
Rank 5 2 4 1 3
Based on the ranking of Table 8 above, frequency and CaCl2 (%) are the least important for
signal-to-noise.
7.3.4. MICROCAPSULE SPHERICITY
Figure 20: The plots of the main effects for means of microcapsule sphericity.

Table 9: Summary of the main effects for microcapsule sphericity.
1 1.339 1.089 1.151 1.092 1.237
2 1.149 1.279 1.159 1.204 1.166
3 1.171 1.255 1.341 1.3 1.315
4 1.107 1.307 1.195 1.224 1.141
Delta 0.232 0.218 0.19 0.208 0.174
Rank 1 2 4 3 5
Figure 21: Main effects plot for signal-to-noise (sphericity).
Table 10: Summary of the S/N ratios and the ranking of the main effects (sphericity).
1 16.69 26.29 19.92 22.85 19.18
2 26.03 18.06 28.67 19.43 21.11
3 19.79 19.51 18.01 24.14 18.49
4 22.39 17.38 20.37 18.93 25.43
Delta 9.34 8.91 10.66 5.21 6.95
Rank 2 3 1 5 4

7.3.5. MICROCAPSULE STRENGTH
Figure 22: The plots of the main effects for means of microcapsule strength.
Table 11: Summary of the main effects for microcapsule strength.
1 0.01012 0.01788 0.02405 0.03193 0.06229
2 0.0527 0.02542 0.02951 0.0729 0.01067
3 0.03201 0.04114 0.06238 0.01222 0.03291
4 0.04517 0.05557 0.02406 0.02295 0.03413
Delta 0.04258 0.03769 0.03833 0.06068 0.05162
Rank 3 5 4 1 2
Figure 23: Main effects plot for signal-to-noise (strength).

Table 12: Summary of the S/N ratios and the ranking of the main effects (strength).
1 -40.8 -39.5 -36.27 -32.49 -29.96
2 -32.02 -34.17 -34.24 -26.57 -40.49
3 -32.98 -29.11 -29.83 -39.15 -30.62
4 -29.17 -32.19 -34.63 -36.77 -33.9
Delta 11.63 10.4 6.44 12.58 10.53
Rank 2 4 5 1 3
7.3.6. PERCENT TRANSMITTANCE OF RECEIVING SOLUTION
Figure 24: The plots of the main effects for means of percent transmittance.
Table 13: Summary of the main effects for percent transmittance.
1 96.46 99.28 50.32 49.72 65.47
2 96.07 66.53 49.78 72.49 73.11
3 42.67 48.94 72.65 66.67 74.18
4 26.36 46.82 88.81 72.68 48.8
Delta 70.1 52.46 39.03 22.97 25.39
Rank 1 2 3 5 4

Figure 25: Main effects plot for signal-to-noise (percent transmittance).
Table 14: Summary of the S/N ratios and the ranking of the main effects (percent transmittance).
1 39.68 39.94 22.23 22.89 30.11
2 39.65 31.05 22.05 30.83 30.97
3 21.28 22.04 31.71 30.28 30.92
4 14.26 21.85 38.88 30.87 22.87
Delta 25.42 18.09 16.83 7.98 8.1
Rank 1 2 3 5 4
7.3.7. SYNOPSIS AND RECOMMENDED LEVELS
Following the one response at a time analysis, the results for factor have been merged in
order to recommend a set of “combined” settings to optimize all responses (Refer to the Tables
below). MINITAB Version 15 does not have the capability to predict the optimum levels for
Taguchi Designs.

Figure 26: DOE Analysis and Methodology for interpretation for % CaCl2 (w/v)
Figure 27: DOE Analysis and Methodology for interpretation for % Chitosan (w/v)

Figure 28: DOE Analysis and Methodology for interpretation for Frequency (Hz)
Figure 29: DOE Analysis and Methodology for interpretation for Voltage (V)

Figure 30: DOE Analysis and Methodology for interpretation for Distance (mm)
The final optimized process parameter derived were CaCl2(10% (w/v)), Chitosan (0.25% (w/v)),
Frequency(600 Hz), Voltage (35V) and 3mm (distance being treated as dummy final outcome
due to manual DOE setup)
Figure 31: Outcome of the DOE analysis- L16 Taguchi Method (MINITAB Analysis): Optimized
Process Window.

8. CONFIRMATION OF COMBINED AND OPTIMIZED PROCESS PARAMETERS
With the optimized process parameters –CaCl2 (10% (w/v)), Chitosan (0.25% (w/v)),
Frequency (600 Hz), Voltage (35V), Distance (3mm) the jetting experiment was re-performed to
confirm the results. Blue Dextran 500 KDa was used for capsulation. The image below shows
the result of capsulation with blue dextran.
Figure 32: Microscopic analysis of capsules with blue dextran run with optimized parameter.
During the jetting process due to stabilization issue with print head (meniscus stability)
except Spheroicity (not perfectly circular microcapsules) the other parameters: Size, Strength and
Turbidity were confirmed. The strength was confirmed with the help of a centrifuge (Relative
Centrifugal Force-RCF is 41760). Figure 33 represents a successful example of circular
microcapsules with the optimized process parameters.

Figure 33: Microscopic analysis of capsules with optimized parameter.
9. QUALITY IMPROVEMENT PROPOSALS
9.1.COVER DESIGN
The following was the proposed cover design to ensure the quality encapsulants. The
material proposed for the design is Acrylic DR101 (Clear). Figure 34 and Figure 35 Provide
details to the design.

Figure 34: Cover Design for bioinkjet printer
Figure 35: Latch and Hinge Specification

Figure 36: Image of Cover Design with Bioinkjet printer.
9.2.DISTANCE MEASUREMENT TECHNIQUE
It seems that there is a need for accurate distance measurement, from the tip of the print
head to the receiving solution. A proposed approach is to install a measuring device to the stage.
A caliper fixture to the base of the stage at one end and to the moving stage (top section) at the
other can be used to measure how far the stage moves (see figure below). Once the receiving
solution is ready and placed onto the stage the stage is moved upto the tip of the print head (be
mindful not to make physical contact between the print-head and the receiving solution). The
caliper is then reset to zero and then as the stage is moved away from the print head an accurate
distance is obtained. The total cost of this implementation is approximately $60.

Figure 37: Caliper device for measuring distance between the print-head tip and the receiving
solution.
10. ECONOMIC JUSTIFICATION
10.1. EXECUTIVE SUMMARY
PROBLEM STATEMENT
 Product 1: Regenerative medicine using encapsulation technique is seen as a perfect
model (hybrid model- biomedical devices and pharmaceutical industry) solving many
of issues related to diabetes, artificial organs, cancer etc. The primary
equipment/technology used in encapsulation technique is the bioinkjet printer. There

are two essential features that make up a bioinkjet printer—the bioink and the inkjet
printer. The encapsulant material (bio-ink) that is used is alginate. Alginate is used
because they show low to no toxicity and low immunogenicity while providing good
biocompatibility and low cost. Optimal print head parameters are essential to control
the size, strength and shape of each droplet (encapsulation). If the encapsulants are
weak or not round it shape it might lead to application issues like exposing the
encapsulating material to White Blood Corpuscles or Human immunity causing issues
with the application
 Product 2: In 2010 there were close to 181 million people affected by diabetes and
kidney failure. Treatments for those affected are in the form of biomedical devices
that needs to be monitored on a periodic basis or having constant dosage of drugs in a
timely fashion to keep the body functional. This not only reduces the overall quality
of life, but also makes it expensive for people affected to keep the treatment going.
SOLUTION AND VALUE PROPOSITION
 Product 1: The focus of the study is to determine the feasibility of obtaining a
universal parameter for multiple print heads and thereby increasing the yield and
overall process efficiency for encapsulation. Taguchi Design of Experiments was
performed to achieve this goal.
 Product 2: Combining both biomedical devices and pharmaceutical industry it not
only provides a cheaper solution but also enhances the quality of life for an affected
individual (no need for monitoring or any intervention in day to day lives).

APPROACH TO MARKET
A unique approach called ‘TIC-TOK’ is conceived for this project to ensure a
profitable business and faster investment returns. Process optimization and yield
improvement will enhance yield with respect to encapsulation research, thereby enabling
quicker return on investment. Product 1 (TIC) is selling a bioink printer with optimized
process recipe. The money from Product-A (TIC) can be funneled towards Product-2
(TOK) that will enable to use the same encapsulation techniques to build artificial organs
targeting a bigger market (101.5 billion dollar market-annual).
CUSTOMERS
 Product 1 (TIC) targets research type environments like universities and small
scale business application.
 Product 2 (TOK) targets providing solution for diabetes and renal failure industry
– both biomedical and pharmacy industries.
MARKET SIZE
 Product-1 has a market size of 5 million dollars (annually).
 Product-2 has a market size of 101.5 billion dollars (annually)
COMPETITORS
 Product 1: Major competitor is San Diego based startup called Organovo.
 Product 2: is targeting both Biomedical Devices Market and Pharmaceutical
Industry related to diabetes industry its keep competitors include Johnson &
Johnson, Bayer, Abott Lab, Roche, Medtronic & Novo Nordisk, Eli Lilly, Sanofi-
Aventis & Sooil.

REQUIRED CAPITAL
 Product 1: Development and implementation cost comprises of equipment cost,
material cost and labor hours. The total estimate cost of equipment, materials,
and labor is $785,164.
 Product 2: Development and implementation cost comprises of FDA application,
equipment cost, material cost and labor hours. The total estimate cost of FDA
application, equipment, materials, and labor is $1.2 million dollars.
BREAKEVEN POINT
 Product 1: The breakeven occurs at 2012 (After 2 years of product launch)
 Product 2: The breakeven occurs at 2021 (After 2 years of product launch)
REVENUES AFTER 3 TO 4 YEARS
 Product 1: After 3 to 4 years of product launch the expected revenue from
product 1 is between 1.6 to 1.8 million dollars.
 Product 2: After 3 to 4 years of product launch the expected revenue from product
2 is 13.3 to 14.6 million dollars.
10.2. PROBLEM STATEMENT (DETAILED)
Product 1: Regenerative medicine using encapsulation technique is seen as a perfect model
(hybrid model- biomedical devices and pharmaceutical industry) solving many of issues related
to diabetes, artificial organs, cancer etc. The primary equipment/technology used in
encapsulation technique is the bioinkjet printer. There are two essential features that make up a
bioinkjet printer—the bioink and the inkjet printer. The encapsulant material (bio-ink) that is
used is alginate. Alginate is used because they show low to no toxicity and low immunogenicity
while providing good biocompatibility and low cost. The bioinkjet printer is a piezo driven

devices where the drop size is controlled by the waveform sent to the print head. Print head
parameters are essential to control the size, and shape of each droplet (encapsulation). These
parameters (factory parameter) are unique to each print heads. There is a initial setup time
required for each print head. This increases the setup time and decreases overall
yield/encapsulation process time. Thus reducing the research efficiency to create an perfect
encapsulation solution.
Product 2: The patients suffering from diabetes have to constantly look for their insulin level
to ensure their sugar level doesn’t go up. This is done by taking right medication through drugs
or feeling insulin to the body through a biomedical device (insulin pump). This hampers there
day to day activity and makes it less normal. Product 2 is intended to create a solution to marry
biomedical device and drug so that it can be embedded into the patient thus reducing their
dependency on devices or drugs and bring back a normal life.
10.3. SOLUTION AND VALUE PROPOSITION (DETAILED)
Product 1: The focus of the product is to determine the feasibility of obtaining an optimal
parameter to enhance the strength and quality of the encapsulants. Taguchi Design of
Experiments was performed to achieve this goal. As this product is not considered to be a
medical device it is not heavily regulated by FDA which means reduced risk and faster return of
investment to the investors.
Product 2: Combining both biomedical devices and pharmaceutical industry it not only
provides a cheaper solution for the patients but also enhances the patients quality of life. Some
of the key value proposition for product 2 being:
o Combines both drug & Insulin & glucose market into one solution
o No need to keep monitoring

o Less external environment intervention in day to day life. Improvement of
quality of life (pseudo artificial pancreas or kidneys)
o Cheaper Mass production
o Saving of 60-88% saving over traditional drug or biomedical solution.
10.4. MARKET SIZE (DETAILED)
Product 1 has a market size of 5 million dollars (annually). Product 2 has a market size of
101.5 billion dollars (annually) i.e. Regenerative medicine using encapsulation technique is
targeted to replace both drug and Insulin & glucose market. i.e. targeting 27% of diabetes :
101.52 billion dollar market for 2010.
10.5. COMPETITORS AND MARKET GROWTH (DETAILED)
Product 1: Optimized Printhead Bioinkjet printer.
Since this is a new market, there are very few competitors such as Inventec, Giltech and
Organovo [54,55,56]. Of these competitors the only company that is actually out in the market
and selling Bioprinters is Organovo [57]. They manufacture and sell a bioinkjet printer called
“NovoGen MMX Bioprinter”. Organovo has a staff of 1-25 people and had a sales of 0.75
million [58] for 2010 with an expected growth of 10-15% year over year and market of 75
Million USD [59]. Thus just going with the projected market growth of Organovo it might be
safe to assume a market growth of 10-15% year over year.

Product 2 : Insulin stent (encapsulation solution)
Regenerative medicine using encapsulation is focused on replacing drug & biomedical
devices (insulin pump & glucose monitoring and dialysis) market the following competitors are
major players per the treatment type:-
 Drugs
o Major players: Johnson & Johnson, Bayer, Abott Lab, Roche, Medtronic &
Novo Nordisk, Eli Lilly, Sanofi-Aventis & Sooil
o Market for 2010 for this segment was 34.95 billion USD[60].
o Growth: 6-10%
 Biomedical Devices : Insulin pump, Glucose monitoring & Dialysis
o Major players: Novartis, Glaxo, Mercks, Pfizer, Eli Lilly and Amylin
o Market for 2010 for this segment was billion 66.58 USD
o Growth: 10%
Thus assuming that encapsulation will be capturing both the drug and biomedical device
market it would be safe to assume a year over year growth of 8-10% (conservative number) once
FDA clearance is through.

10.6. BUSINESS MODEL (DETAILED)
The proposed business strategy is a two phase approach (TIC-TOK approach).
Figure 38: Approach to Market
As shown in Figure 38 above, under this approach the company will launch two primary types of
product and services under two phases of business development. The first phase is dedicated to
the successful launch and marketing of the bioinkjet printer and its consumables. The capital
equipment is considered to be the bioinkjet printer system as a whole, and the consumables are
the bioinkjet printer’s print-heads and bioink. The print-head and bioink are paired together
because this will ensure optimal quality of encapsulates and high throughput. Product 1 is
expected to have a quick development and quick return of investment. The revenue generated
from Product 1 will be invested into the development and launch of Product 2.
Phase two of the business development will be focused on creating a combination device
(Product 2) using stents and encapsulated insulin producing beta cells. Successful release of

Product 2 will be both time consuming and expensive project. This is why the revenue of
Product 1 is needed to support the development of Product 2.
10.7. COST (DETAILED)
10.7.1. PRODUCT 1 - DEVELOPMENT AND IMPLEMENTATION COST
Development and implementation cost comprises of equipment cost, material cost and labor
hours. As the production increases, so do the materials and staff of the organization. This project
requires the following materials and equipment, provided by the Bioengineering Laboratory at
San Jose State University. The costs of equipment and materials required by the experiment are
listed in Table 15 and Table 16. Therefore, the total estimate cost of equipment, materials, and
labor is $785,164. All equipment is purchased as a one-time cost. Since the equipment can be
used for an ongoing period of time, it will not be considered as operating costs but rather as
standard start-up costs.
Table 15: Costs of equipment
Equipment Cost Type Total Cost
Incubator One time 1,295
Autoclave One time 6,000
Inkjet Printer One time 10,000
Centrifuge One time 3,000
Temperature Controlled Shaker One time 2,000
Microscope One time 15,000
Biological Safety Cabinet One time 6,000
Scale One-time 1,500
Needles One time 200
Computer (Cell counting, QC, inkjet,) One-time 30,000
Video Capture Card One time 4,000
MicroFab Jet Lab II Subsystems Print head One time 3,000
C-03 reservoir caps One time 75
Waveform Control Software One time 8000
Lab VIEW Software One time 59.95

Aphelion Drop Analysis Software One time 8,000
Total $98,130
Table 16: Cost of materials
Material
Cost Per
Quarter
Total
Cost
Supplies and Disposables (Autoclave
bottle, gloves, disposal bags, beaker,
cylinder and so on)
3,000 12,000
Automatic Pipettes 3,000 12,000
Microencapsulation Materials 15,000 60,000
Nitrogen 1,200 4,800
25mm Syringe filters, 5 μm depth Filter,
nylon for aqueous fluids
225 900
Islet Cell 4,000
Total $22,425 $93,700
At different development stages, the company needs to hire different specialists such as cell
culture engineer, device engineer, to perform necessary tasks (Table 17). These personnel will
function to provide effective products and to make sure these products can be marketed to the
public.
Table 17: Labor cost
Staff
Experience
(years)
# People
Cost
(per month)
Total
Cell culture
engineer
PHD(>2) 2 $19,445 233334
Regulatory
FDA
>3 1 $4,167 $50,000
Device
Engineer
> 3 1 $5,000 $60,000
QC engineer
(quality
control)
0-1 1 $4,167 50000

CEO/
President
1 $16,667 200000
Total $49,446 $593,334
10.7.2. PRODUCT 1 – OPERATING EXPENSES & BREAKEVEN POINT
Fixed costs include the staff and equipment. All equipment is purchased as a one-time cost
and each employee will earn a 5% raise. Variable costs are the materials. The variable costs are
forecasted to rise by 10% each year as we continue to increase our customer base. Product 1 will
be sold for $138,686 which is a competitively priced under the competitions. Ongoing revenue
is generated thru sale of consumables and or annual fee of intellectual property price for software
($602). The revenue estimate (Table 18) is based on a conservative forecast that approximately
10 products will be sold during first year. Assuming the loss from each year carries over to the
next year, the profit from each subsequent year makes up for the initial loss. The Breakeven
Analysis is seen in Table 18, where the cost of production and revenue from the product are
analyzed. The revenue and expense chart is seen in Figure 39.
Table 18: Total cost breakdown and breakeven analysis
Year Fixed Cost Variable Cost Total Cost Revenue Loss & Profit
2011 691464.0 93700.0 785164.0 0.0 -785164.0
2012 691464.0 93700.0 785164.0 1386873.0 -183455.0
2013 623000.7 103070.0 726070.7 1531584.8 805514.1
2014 654150.7 113377.0 767527.7 1676899.1 909371.3
2015 686858.3 124714.7 811573.0 1822815.7 1011242.8
2016 721201.2 137186.2 858387.4 1969334.9 1110947.5
2017 757261.2 150904.8 908166.0 2116456.4 1208190.4
2018 795124.3 165995.3 961119.6 2264180.5 1303060.9
2019 834880.5 182594.8 1017475.3 2412506.9 1395031.6
2020 876624.5 200854.3 1077478.8 2561435.9 1483957.0
2021 920455.8 220939.7 1141395.5 2710967.2 1569571.1

Figure 39: Revenue (Income) and expense chart for 2011 to 2021
Figure 40 is the chart for breakeven analysis. The company will breakeven in 2012 and
continue to profit.
Figure 40: Net Profit/Loss Chart per Year
-2500000.0
-1500000.0
-500000.0
500000.0
1500000.0
2500000.0
2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021
Revenue
Variable Cost
Fixed Cost

10.7.3. PRODUCT 2 - FDA AND CLINICAL TRAILS
If a device is classified as a novel drug a clinical trial will most likely be required for a
premarket approval. The FDA defines drug as following: If the primary intended use of the
product is achieved through chemical action or by being metabolized by the body, the product is
usually determined to be a drug (definition from CFR). Microencapsulated products, such as the
Insulin stent, fall within this definition and must therefore go through the long FDA approval
process. Typically a clinical trial consists of five phase and the first three are needed for FDA
approval. The outline below summarizes these phases [61
]:
 Phase I
Researchers test a new drug or treatment in a small group of people (20-80) for the first
time to test its safety, identify the maximum tolerated dose, find a safe dosage range and
identify side effects.
 Phase II
The drug or treatment is given to a larger group of people (100-300) to see if it is
effective, to further evaluate its safety and to gather additional information regarding safe
dose range.
 Phase III
The drug or treatment is given to large groups of people (1,000-3,000) to confirm its
effectiveness, monitor side effects, compare it to commonly used treatments and collect
information that will allow the drug or treatment to be used safely.
 Phase IV
During this phase, investigators are looking for additional information, including the drug

or treatment's risks, benefits, and optimal use. This trial may occur after the drug or
treatment has been approved for use by the FDA.
 Phase V
Trials may be conducted to determine better dosing guidelines, new formulations, effects
on different populations or new indications.
Prior to conducting Clinical trials the device must be tested to assure that it is reasonably safe
for initial use in humans and that the device’s performance justifies commercial development.
The device will need to be cleared as an Investigational New Drug (IND) status in order for it to
be exempt from legal requirements and used in clinical trials. The IND application must contain
information in three broad areas:
 Animal Pharmacology and Toxicology Studies - Preclinical data to permit an assessment
as to whether the product is reasonably safe for initial testing in humans. Also included
are any previous experiences with the drug in humans (often foreign use).
 Manufacturing Information - Information pertaining to the composition, manufacturer,
stability, and controls used for manufacturing the drug substance and the drug
product. This information is assessed to ensure that the company can adequately produce
and supply consistent batches of the drug.
 Clinical Protocols and Investigator Information - Detailed protocols for proposed clinical
studies to assess whether the initial-phase trials will expose subjects to unnecessary
risks. Also, information on the qualifications of clinical investigators--professionals
(generally physicians) who oversee the administration of the experimental compound--to
assess whether they are qualified to fulfill their clinical trial duties. Finally,
commitments to obtain informed consent from the research subjects, to obtain review of

the study by an institutional review board (IRB), and to adhere to the investigational new
drug regulations.
Once the IND is submitted, the sponsor must wait 30 calendar days before initiating any
clinical trials. During this time, FDA has an opportunity to review the IND for safety to assure
that research subjects will not be subjected to unreasonable risk [62
]. Clinical trials for new drugs
are both costly and time consuming. According to GCP for Medical Device Trials, an approval
for marketing devices in the US follows in five to ten years, then an additional five to ten years
for Japan, which has the longest regulatory pathway. A medical device clinical trial can cost
between $5 and $10 million in the United States or Western Europe and more in Japan. The cost
of the same trial conducted in Eastern Europe will be considerably lower, and in India, China, or
Korea, it may be 1/10 as expensive [63
]. Furthermore, additional cost that should be taken into
consideration is the FDA application cost for new drug. The table below is an estimated cost
associated with FDA application fee, assuming that the application process starts in 2012. This
estimate is based on “the Prescription Drug User Fee Act”. The average time is 5 years with an
expected application cost of $3.8 million.
Table 19: FDA application fee [64
]
Year Submission Type
Standard
Cost
Number of
submissions
Estimated
FDA Cost
2012 Manufacturing Supplement–CDER $6,000 3 $18,000
2012 Manufacturing Supplement–CDER $34,000 1 $34,000
2014 New Drug Application–New Molecular Entity $1,243,000 1 $1,243,000
2015 Investigational New Drug Application–CBER $266,000 2 $532,000
2015 Supplement with Clinical Data–CDER $210,000 1 $210,000
2016 Product License Application–CBER $1,194,000 1 $1,194,000
2016 Establishment License Application–CBER $622,000 1 $622,000
Total $3,853,000

10.7.4. PRODUCT 2 - COST OF MICROENCAPSULATION
Typically a patient receives at least 10,000 islet cells per kilogram of body weight. The
alginate was mixed with cultured islet cells to a concentration of 2,000 islets/ml [65
]. Table 20
shows the cost to run one experiment and make 10ml of microcapsules is $402.04.
Table 20: Cost of each batch of microencapsulation
Materials Amount/Batch Batches
Final Price
($)
Price/ Batch
($)
Sodium Chloride 9g/L 111 60.1 0.54
Calcium Chloride 15g/L 100 107.1 1.07
Medium molecular weight
sodium-alginate
0.75g/50ml 133 34.8 0.26
Low molecular weight
sodium-alginate
50mg/50ml 2000 38.9 0.02
Sodium citrate dihydrate 0.81g/50ml 370 54 0.15
Islet Cell 1 batches 10 4000 400
Total/Batch $402.04
10.7.5. PRODUCT 2 - OPERATING EXPENSES & BREAKEVEN POINT
The cost estimate and breakeven point of Product 2 is a continuation of cost analysis done by
the previous team working on the same project. The calculations are based on an implantable
device, Insulin Stent, which is intended to treat Diabetic patents. The first Table indicates each
year’s cost for the company from the time of initial production to 7 years into production (the
first two year includes development of the manufacturing facility).
There are two types of cost taken into consideration—fixed and variable cost. Costs
associated with fixed costs include labor cost, rent, and other non-recurring costs. Whereas, costs
classified as variable costs are the costs directly associated with amount of product
manufactured. It is assumed that once Product 1 starts making profit in 2013, half of its revenue

will be funneled toward supporting the development of Product 2. The breakeven analysis is
calculated under the assumption that the clinical trial and FDA regulation will go on for no more
than five years. Therefore, it is estimated that the product will break even in the 3 year of its
market launch (Market launch of 2019 and break even at 2021) with market share of 0.01% to
start with and 10% market growth year over year.
Table 21: Total cost breakdown and break even analysis.

Figure 41: Revenue (Income) and expense chart for 2011 to 2021
Figure 42 is the chart for breakeven analysis. The company will breakeven in 2021 (3 years
into market) and continue to profit.
($5,000,000)
$0
$5,000,000
$10,000,000
$15,000,000
$20,000,000
Revenue
Variable Cost
Fix Cost

Figure 42: Net Profit/Loss Chart per Year
10.8. RETURN OF INVESTMENT (ROI)
 Product 1: After 5 years of product launch the Return of Investment for product 1 is 1.5 with
an initial investment of $785,164.
 Product 2: After 5 years of product launch the Return of Investment for product 2 is 10 with
an initial investment of $1.2 million dollars.
10.9. SWOT ANALYSIS
The proposed business plan is strong as shown in the summary of SWOT analysis below.
The business’s weaknesses are minor, strengths are valuable, Opportunities are many and
threats are manageable.

10.10. EXIT STRATEGY
There are two exit strategies proposed for the products. The first exit strategy for Product 1 is
being bought over by potential buyers from drug industry and for Product 2 is starting with a
joint collaboration with multinational company (biomedical or drug industry) and then
eventually be bought over by the same company.
The second strategy is to work our way through the product life cycle for both product 1 and
2. The following six assumptions are made for the product life cycle:
1. First customer shipment for product 1 to be one to two years from current time.
2. 10 years of product life (for both product 1 and 2) once it hits first customer shipment
(FCS).
3. No FDA application required for product 1

4. Development for product 2 will start at no later time than when product 1 hits the market
(2012).
5. Product 2 to be TYPE 3 Biomedical device and hence expect 7 years for FDA application
and clinical trials before we hit FCS
At this stage for product 1 we already have an Execute commit with an ideal prototype. We
are working on automating the stage which will help improve the reproducibility for quality
samples to help speed up the research market. As mentioned in the approach to market, once we
hit the market for product 1, the revenue from product 1 may be used towards product 2
development, FDA application and clinical trials to help capture the multibillion dollar market.
Product 1- Product Life Cycle Chart
Product 2- Product Life Cycle Chart

Figure 43: Product 1 and 2 product life cycle charts.
11. PROJECT DEVELOPMENT SCHEDULE
The focus of the project development will be on determination of optimal bioinkjet setting.
Determination of the optimal setting can immediately help move the bioinkjet printer toward
application for combating regenerative medicine by reducing the research development time and
cost necessary to investigate and produce the desired microcapsule characteristics. With high
throughput, consistency, and quality of the produced encapsulates, the researchers can
investigate various formulations of the bio-membrane more efficiently. The research progress is
shown in Gantt chart (Figure 44).

Figure 44: Research Project Schedule.
12. CONCLUSION
Combined and Optimized process parameters were derived using Design of Experiment (L-
16 Taghuchi Method): –CaCl2 (10% (w/v)), Chitosan (0.25% (w/v)), Frequency (600 Hz),
Voltage (35 V), Distance (3 mm). Combined and Optimized process parameters were re-run and
three of the four parameters- Size, Strength and Turbidity were confirmed. Based on the re-runs
results as the blue dextran did not leak it is safe to assume that pore size of microcapsule is less
than 9-10 nm (size of the blue dextran markers). Quality Improvement of the experimental setup
in the form of Cover Design and Distance measurement techniques were proposed.
13. FUTURE DIRECTIONS
With a clean and stable print head and Combined & Optimized Process Parameters a re-run
be performed to confirm the sphereocity. With the proposed mechanical enhancements to the
system, the confirmations runs will be re-visited. A sub-set of the factors will be used for
conducting a factorial design in order to establish a linear model for the sphericity and size

responses. These models will be invaluable for the future applications of bacterial and
mammalian cell encapsulation.

APPENDIX
Turbidity Calculations-

MINITAB run matrix-

 Images from each of the DOE Runs:-
Figure 45: 5% CaCl2 (w/v) -No Chitosan (w/v) -300Hz-30V-1mm
.
Figure 46: 5% CaCl2(w/v) -0.25Chitosan (w/v) -400Hz-32V-2mm
10 um

Figure 47: 5% CaCl2(w/v) -0.5Chitosan(w/v) -500Hz-35V-3mm

Figure 50: 10% CaCl2(w/v) -0.5Chitosa(w/v) -600Hz-30V-2mm

Figure 51: 10% CaCl2(w/v) -0.75Chitosan (w/v) -500Hz-32V-1mm
Figure 52: 15CaCl2(w/v) -0.75Chitosan(w/v) -400Hz-30V-3mm

Figure 55: 15% CaCl2(w/v) -No Chotsan(w/v) -500Hz-37V-2mm
Figure 56: 20% CaCl2(w/v) -0.25 Chitosan(w/v) -500Hz-30V-4mm

Figure 57: 20% CaCl2(w/v) -0.5%Chitosan(w/v) -400Hz-37V-1mm
Figure 58: 20%CaCl2(w/v) -0.75%Chitosan(w/v) -300Hz-35V-2mm

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