After having treated thousands of patients with debilitating dry eye, I and my team are looking to the future. 

We are excited about what the future has in store as we are close to finding a better way to treat dry eyes and ideally cure dry eyes and meibomian gland disease. 

Here is where we are:

1. Cord Blood Serum: we are using this and it is working to relieve symptoms. The first 2 patients we treated were shocked it worked so well. The first patient who has traumatic neuropathy said it helped significantly, better than anything else she has tried, and she is going to start a second round. But it was not a cure; she still has some pain when not using the Cord Blood Serum.

2. Cord Blood Serum + Platelets + Autologous Stem Cells: this might be our best chance to regrow meibomian glands and heal mucin and lacrimal glands cells. 

3. Oxervate; have 2 patients about to try this who have severe neuropathy. We are hopeful this will help. I am eager to do a trial of PRP drops in 1 eye and Oxervate in the other as I think PRP works better but no pharmaceutical company would fund such a trial. 

3. 3D printed conjunctiva, mucin glands, meibomian glands, lacrimal glands: this is the future. We are still far away but we are working on this already. We are looking into this also to help our patients who have a pterygium.

4. Implantable glands secrete the perfect tear with every blink: patent pending. 

5. Perfect Oil Reservoir: a reservoir with a little tube that pumps oil into the inferior fornix. It will attach to a pair of glasses so less noticeable and painful: patent pending. 

Currently we are applying for research grants to fund our research. If you or anyone you know is interested in helping us raise money for these research projects, please contact Kim Neal at KNeal@voeyedr.com.

References:

1. 3D Printing of Tissue

Human Organ and Tissue Engineering: Advances and Challenges
in Addressing the Medical Crisis of the 21st century
Melanie P. Matheu, PhD
Erik Busby, PhD
Johan Borglin, PhD

 ABSTRACT
Organ failure is the imminent health-care crisis of the 21st century. In the United States, the estimated
cost of lung, kidney, and liver disease combined is upwards of $256B annually.
1, 2, 3 One in seven adult
Americans is suffering from some form of progressive kidney disease, and currently, 660,000 adults in the
US are suffering from end-stage renal failure.4, 5, 6 Lung disease is the 3rd leading cause of death,
7 liver
disease is the 12th leading cause of death,
4 and heart disease, the number one cause of death in the US,
can be caused, or exacerbated by, loss of organ function.7, 8 Endocrine diseases can also be thought of as
a failure in an organ system; a notable example is Diabetes. Globally, Type 2 Diabetes affects over 400
million people,
9 while Type 1 Diabetes affects fewer patients, the numbers are in the millions. Type 1
Diabetes is autoimmune-driven and has an annual global increase in incidence of 10 percent year over
year.
10, 11 Both Type 1 and Type 2 Diabetes can benefit from transplantation of tissue known as the Islets
of Langerhans, an essentially curative procedure that leads independence from insulin injections.
12, 13
Transplantation is the only known curative option to address organ failure. Development of vascularized
organs and tissues for transplantation is not feasible with current technology. Despite the development
of numerous methods, none have broached what will ultimately become a multi-trillion-dollar global
market that can address hundreds of millions of patient’s needs: living human organ and tissue
replacement. Here, we review current approaches in the race to produce functional human organs for
transplantation, discuss roadblocks, and present novel technology that will pave the way to building
human organs in the laboratory setting.
Author Affiliations:
Founder, CEO Prellis Biologics, Inc. (MPM), Principle Optical Engineer (EB), Principle Optical Engineer (JB) 

INTRODUCTION
Organ and tissue transplantation can restore physiologic homeostasis, reduce stress on other organ
systems, and improve the lives of the recipients. Donor-derived transplantation, however, is significantly
limited by supply, with only 34,771 organ transplants being performed in the US in 2017, and well over a
million potential recipients turned away from a waitlist, or waiting for an immunologically matched
donor.
14 Efforts in the regenerative medicine space have advanced treatment options, and include work
with patient-derived stem cells to promote tissue repair, as well as development of improved
immunosuppression options. However, medical options to treat end-stage organ failure have not
advanced for nearly 50 years, and still require human-to-human donation. This leads to a limited number
of healthy organs available for transplantation and only select candidates are added to the waitlist to
receive a life-saving organ. Tremendous advances in regenerative medicine have been reported in the last
decade, but in terms of human organ engineering, the field remains stalled at thin sheets of cells that lack
capillary blood flow, and therefore are not suitable transplantation.
In the United States, an estimated 150 million people are suffering from kidney, lung, or liver disease, the
3rd, 9th, and 12th leading causes of death, respectively.
15, 16, 17 Co-morbidities including heart failure precede
palliative care, often for decades, taking a significant toll on patients, their families, and the health care
system.1, 8, 18, 19 Compounding the toll on our healthcare system, it has recently been reported that
extended periods of time with low organ function are associated with progressive loss of cognitive
function and physical abilities.20, 21 Even in a reasonably well-managed disease such as Diabetes, it is
reported that autonomic and central nervous system decline is accelerated, leading to increased incidence
of dementia, cognitive decline, as well as an increased risk of heart attack and renal failure.22, 23, 24 As we
age, decline in organ systems may contribute to and precipitate neurological disorders, or induce
significant stress on other organ systems including the heart.21, 25, 26, 27
Organ transplantation allows patients to reclaim normalcy, return to work, and in many cases, extends
life by decades. However, lifesaving transplantation is limited by too few viable organs. Donation from
16,468 deceased individuals led to a record-breaking 34,771 transplants performed in 2017 (more than
one organ can be harvested per donor).
14 The increase in healthy organs for transplantation is widely
attributed to the current opiate addiction crisis,
28 and does not represent a sustained increase in donation
which, until 2015, remained below 30,000 transplant surgeries per year in the US.
29 Typically, fewer than
16,000 organ donors give the gift of life each year, far fewer than the millions of patients who could benefit
from an organ transplant.
Although significant efforts have been made to bridge the chasm between organ availability and need,
increasing organ donor numbers and improving organ procurement procedures will not bridge the gap.
Advances in stem cell biology and novel engineering approaches have brought scientists closer than ever
before in medical history to producing a functional replacement human organ. The race between three
technologies in the field will likely bring the capability to produce fully functional replacement human
organs into medical reality: recellularization of tissue structures, xenograft transplantation (from animals),
and 3D printing to manufacture organs. Here, we focus on the field of organ and tissue development in
the laboratory setting using 3D printing, and provide a brief overview of other technologies, including
recellularization, and the efforts to develop porcine breed-stock for xenograph transplantation.

 MANUFACTURING 3D PRINTED HUMAN ORGANS AND TISSUES
There are four requirements for scalable 3D printing of functional human organs: Resolution, Speed,
Complexity, and Biocompatibility. 

RESOLUTION
High resolution printing is critical for building single-cell walled capillaries and microcellular niches,
necessary components of functional organs and tissues.
All living tissues and organs are perfused by 5-10 micron (in diameter) blood vessels that are known as
capillaries. Capillaries are the smallest blood vessels in the body and serve as the primary exchange point
for oxygen, nutrients, and cellular wastes. After nutrient and waste exchange capillaries recombine to
form the veins that ferry blood back to the heart and then lungs. These small blood vessels are the critical
functional unit in all tissues. Without a network of capillaries, tissues and organs starve for oxygen
(hypoxia), and cells begin to die off in a matter of minutes leading to tissue necrosis (death). Therefore,
the resolution of a given printing process must be about or near that of a single cell.
Organs need oxygen for cellular respiration, but also require thin-walled capillaries to function effectively.
Within many organs, capillaries play additional functional roles. Kidneys, for example, require single-cell
walled capillaries for blood filtration, lungs require single-cell walled capillaries for gas exchange, and the
liver requires single-cell walled capillaries for waste processing. The functional networks of capillaries in
an organ are extensive. A human kidney contains 12 miles of glomerular capillaries that are precisely
placed to filter blood and reabsorb salts and nutrients.30 The human brain, which uses 20% of our inhaled
oxygen, boasts over 400 miles of capillaries, where each neuron has a dedicated capillary.31, 32
Living tissue dependence on capillaries makes bioprinting at a high resolution a critical element in 3D
printing and manufacturing of organs. Although large structures can be created with current 3D
bioprinting technologies, the smallest blood vessels created to date are on the order of 50 to 1,500
microns in diameter, too large to act as functional capillary networks.33, 34, 35, 36, 37 The average inner
diameter of a capillary is 5 to 10 microns, about one one-thousandth of a centimeter. This inner diameter
is so small, red blood cells pass through in single file. In organs and tissues, capillaries are spaced a
maximum of 250-300 microns apart, the limit of diffusion for oxygen (O2). Capillary spacing can vary
significantly, and in tissues with high metabolic requirements, such as cardiac tissues, capillaries are much
closer together, an average distance of 20 microns apart.
38, 39 Therefore, a printing technology must
achieve both fine enough resolution to create a thin-walled capillary, while allowing for cells to be placed
between capillaries. Without the ability to build a functional vascular system, human tissue engineering
will not progress beyond thin sheets of cells.
40 The lowest reported resolution to date was achieved with
two-photon raster-scanning of a laser.
41 Spray-based deposition printing can print the lowest resolution
of the non-light based systems and allows for 50 micron resolution, still 5 to 10 fold to large to build
functional tissues.42
Along with building vasculature, the ability to place single cells allows for the creation of microcellular
niches, an important aspect of tissue engineering. Microcellular niches are small groupings of diverse cell
types that create a self-sustaining, highly specialized micro-environment through chemical cross-talk and
cell-cell interactions. Often, as with the crypts of the small intestine, these niches contain stem cells to
replenish local cell populations during tissue remodeling, repair, and during the natural course of cell
death.
43 In vitro cells can be induced to self-organize to create these niches, however often fail to achieve
a fully organized system, this may be due to geometries and intracellular signaling factors.
44, 45, 46 However,
the capacity for cells to organize and differentiated once placed in a larger tissue with circulation remains
to be determined. It is likely that cells will have to have specific 3D organization to recapitulate functional
microcellular niches. Therefore, the ability to place groups of cells with high resolution is likely to be an
important component of tissue and organ engineering.
In sum, the physiologic requirements for organ structure and function require single cell placement to
build capillaries and microcellular niches. Therefore, the resolution for creating tissues, organs, and
extracellular matrix should fall within the range of a single cell, between 1 to 10 microns.
SPEED
Unlike a standard manufacturing process, human tissue engineering is constrained by the finite lifetime
and health of the cells being used to create the tissue.
Complex organs contain cells at various stages of differentiation (or development). Extended periods of
time in a sub-par environment may induce undesirable cell differentiation. While many cell types can
survive in carefully-controlled culture conditions for up to two to three months, high pressure, speeds,
sheer force, and heat created by extrusion or droplet-based printing are not well tolerated by cells.
47, 48,
49, 50
Ideally, printing an extracellular matrix that contains cells would take less than 12 hours before cells are
returned to physiologic conditions; generally 37°C, and 5% CO2 in a nutrient sufficient environment. Cell
death during the tissue printing process is an important consideration as it can result in a ‘dead’ tissue or
significantly alter the environment for the remaining cells.
51 The limit for the percentage of cells that can
be lost during a printing process is unknown, and different cell types have different tolerances for
environmental changes. Extrusion and electrospin based bio-printing causes significant cell death, while
ink jet based bioprinting and other methods have reported higher cell viability.
52, 53 42 Other potential
methods for improving cell viability include cooling of materials, including cryogenic printing which offers
an interesting method for maintain cells. However, the resolution of cryogenic printing is too low to create
vasculature, and defects such as air-bubbles in the range of hundreds of microns in diameter are
reported.
54
To calculate three-dimensional printing times at a given resolution, the type of technology being used to
print must be considered. Three dimensional (3D) light based printing at its fastest described, to date, is
performed through layer-by-layer additive manufacturing through the deposition of a single line or 2D
planar illumination, often via UV-light based excitation.55, 56 UV light is known to be mutagenic, and thus,
despite the demonstration of printing with bioinks that are both cell compatible and UV reactive, repeated
UV light exposure limits the safety of these engineered tissues for use in transplantation. Extrusion
printing of 5-10 micrometer layers has yet to be described and the speed of deposition at these resolutions
is not rapid enough for the creation of microvasculature for transplantable tissues. Therefore, we will
focus primarily on the light-based deposition of structures or photolithography, for assessing speeds of
tissue engineering.
Laser or light-based polymerization of 3D printing materials can print polymerize biological proteins and
soft non-toxic transplantable materials. The polymerization occurs much like the curing process of an
epoxy resin with light. Light can be used outside of the visible range of wavelengths to induce photocrosslinking (Figure 1).
Figure 1. Photo-polymerization of a liquid polymer
This process is both fast and can reach the resolution necessary for single cell layer deposition. Current
laser-based printing approaches utilize a one-dimensional line scan of a pinpoint laser, or 2D planar
deposition to print 3D structures in layers. Scaling of the print volume in 3D leads to significant gains in
speed without compromising resolution. To estimate time required to produce a structure with lightbased polymerization of a material, we must account for both the time required to scan the laser through
the sample and the time that must be spent to polymerize each voxel. A is the voxel cross section
orthogonal to the scan direction, v is the scan velocity, f is the fill factor, �”#$%&’ is the number of voxels
in the print volume, t is the dwell time necessary for material polymerization, and R is the holographic
exposure rate (voxels/s).57, 58 

 Using these equations, we present calculations comparing the printing times of a centimeter cubed with
one micron resolution, a 100 μs dwell time, and two different fill factors (Table 1). Note that polygon
scanners are rate-limited by the dwell time, which leads to fill factor independence. 3D holographic
printing can be scaled by scaling up optical components. The first two numbers are representative of the
fastest technology built by Prellis Biologics prior to introducing custom optical components followed by
the estimates for the next generation system which will print at a rate of 12 million voxels per second.
Table 1. Estimated fastest time to print 1 cm3 with 1 micron resolution
1% fill factor 15% fill factor
Scan speeds (high/low)
Galvanometer (Scanner)
10 mm/s 3.2 years 3.6 years
10 m/s 12.7 days 175 days
Polygon Scanner
10 mm/s (limited by dwell time) 3.2 years 3.2 years
3D Multi-Photon Holography
36,000 voxels/s 3.2 days 48 days
240,000 voxels/s 11.6 hrs 7 days
12 M voxels/s 14 minutes 3.4 hours
Even at 10 mm/s, the resolution is relatively low (10s of microns), and as much as 30-50% what would be
structurally necessary to print is skipped in each illumination pass, severing limiting the cohesive
structures that are constructed at this speed.59 60, 61 3D Multi-Photon Holography developed by Prellis
Biologics, Inc. rapidly prints 3D structures and is described in detail later. Holography is effectively fillfactor independent if sufficient laser power exists. Therefore, in holographic based laser printing speed
not limited by resolution wherein speed is volume dependent, and the print volume is dictated by static
optical components. Holography is achieved with multi-photon excitation through a combination of beam
expansion wave-front shaping which can be thought of as beam-steering that allows for complete or nearcomplete structure deposition (Figure 2).
Figure 2. Single photon absorption as compared to two photon absorption and holographic laser
printing.
Figure 2. Graphics demonstrating laser printing output based on optics of single photon and
multiphoton printing processes and the expected structural outcomes. (A) Graphic depicting
single photon laser projection into a print media containing bath without masking or isolation of
the intended plane of focus, which would be expected to leave a printed structure behind in the
shape of the entire cone of light along the focal length. (B) Graphic depicting a multi-photon
absorption process where the photon density is high enough only at the point of focus, leaving
only a pinpoint structure behind in a photosensitive print media bath. (C) Representative graphic
of wave front shaping to produce a hologram in which the multiphoton absorption process occurs
at multiple points of focus in the x, y, and z planes. In this process, rapid switching between 3D
projected portions of a complete structure may be used to build the complete structure. (D)
Representative graphic showing a complete image projection in multiple planes allowing for
holographic printing of a complex structure.
COMPLEXITY
Biological complexity in 3D organ printing can be defined as the close placement of structural
components that work in concert to carry out the functional purpose of the organ.
For example, the primary functional purpose of the lung is gas exchange between the circulatory system
and the environment. The majority, 90%, of gas exchange occurs across two cells thinly stretched cells in
close association, one that comprises the capillary wall, one that makes up the alveolar cell wall; the endpoint for all branches of the lung.62 Alveolar spaces are structurally similar to a bunch of grapes, where
each alveolus or ‘grape’ is the smallest subunit of lung, measuring a mere 200 microns across. To ferry air
and blood to this membrane, airways divide an average of 23 times between the trachea and the alveoli
where gas exchange occurs. Branched airways and a dual circulatory system create the structural and
physiological support system of the lung which is comprised of 1,500 miles of airways and 700 million
alveolar spaces.
63 The complexity of the lung is not unique: kidney, liver, and even skin each have
structures of similar complexity that are required for physiological function. An engineered tissue must
be able to match the complexity of these functional subunits, structural requirements, and nuances
therein to be considered viable for human donor organ replacement.
Layer-by-layer extrusion printing can achieve some measure of complexity but cannot match the
complexity necessary for creation of human tissue. Short wavelength light, much like that used in resinbased polymerization, can achieve quite a bit of complexity if controlled from multiple point sources;
however, it still lacks biocompatibility and the ability to build true complexity.64 This is reviewed further
in biocompatibility of printing processes.
Multi-photon excitation of a fluorophore, and its use in photolithography, has inherent advantages in that
it yields high complexity. 3D control of structural polymerization and the ability to print behind and inside
of already-printed structures allows for unprecedented complexity. Multi-photon excitation allows for a
3D hologram to be generated with ultra-fine, one-micron resolution, in a mask-less lithography process,
while using 2P long wavelength light that is can pass through already polymerized structures or cells and
be focused on the other side to continue printing. This is achievable because focal plane or voxels in the
case of a holographic projection are the only place where polymerization of the print materials is induced.
Therefore, complexity is easily created by printing behind and even within already existing structures
using multi-photon polymerization.
BIOCOMPATIBILITY
To build an organ or tissue, biocompatibility, is of paramount importance.
First, the printing substrate or materials and process used to create the tissue must be compatible with
living cells. Second, the tissue must be tolerated by the host’s immune system. Third, the materials must
be structurally sound; matching the properties of tissues such that it can operate within the physiologic
requirements of an organ.
Printing materials that are highly biocompatible range from extracellular matrix proteins such as
hyaluronic acid and the most abundant, collagen (60-80% of a given tissue), to biologically inert materials
such as polyethylene glycol (PEG).
65, 66, 67 Collagen is highly evolutionarily conserved wherein human and
animal collagen is often indistinguishable at the biochemical level, leading to cross-species
compatability.
68 Furthermore, collagen is generally immunologically inert with only 2-4% of patients
reacting to cosmetic injections of bovine collagen, a response that can be tested for well ahead of
transplantation.
69 Furthermore, recombinant sources of pure collagen have been developed to bypass any
complications with animal-based production of materials making this an ideal material for the scaffold of
transplantable tissues.
70 Mild cellular toxicity can occur in some printing formulations that include high
amounts of photo initiators; however, with protective formulations and low exposure time, these issues
are not expected to hinder tissue and organ printing via light-induced polymerization.
71, 72, 73
The printing process that creates a cell-containing extracellular matrix must also be biocompatible.
Extrusion and spray-based printing of cell-containing bioinks creates significant sheer force, even at low
resolution, and therefore is not scalable to a higher-speed lower resolution that could maintain cell
viability at the speeds necessary to build whole organs. Light-based polymerization processes do not exert
sheer force on the cells and can print at high resolutions; however, photo-damage and heat-induced
damage can be significant issues. Short-wavelength light in the visible range, projected at a high intensity
is photo-toxic to cells, and UV light sources cause single and double stranded DNA breaks in as many as
10% of the cells with potential to result in oncogenic transformation.
73, 74 Transplantation of a potentially
cancerous cell population would be of prohibitive concern, making UV or short-wavelength light-based
lithography for living bioinks a high-risk proposition.
PRELLIS BIOLOGICS 3D PRINTING TECHNOLOGY
Manufacturing methods have made significant progress towards meeting each of the four requirements
for building human organs from scratch; Resolution, Speed, Complexity, and Biocompatibility. However,
to date, no single technology has been reported to meet all four. Here we present our laser-based
holographic printing process that provides a single platform technology that meets the resolution, speed,
complexity, and biocompatibility, requirements necessary to bioprint human tissues and organs.
By using holographic 3D printing with a far-red laser, we effectively decouple speed from resolution, while
introducing previously impossible complexity. This has allowed, for the first time, the creation of subcellular resolution structures using cell-laden bioink. The applications of this technology are numerous,
ranging from ultra-fast single cell encapsulation to printing of replacement human organs and tissues.
Subcellular Tunable Resolution and Print Areas
In the lithography field, often used for computer chip manufacturing, the resolution obtained by printing
with light can be sub-wavelength (on the order of tens of nanometers). Although the extracellular matrix
that holds tissues and organs together is sub-cellular in resolution, on the order of one-hundredth of a
human hair, these structures are far larger than the components of a silicon wafer, ranging from 200
nanometers to several microns in diameter.
At Prellis Biologics, we have built a photo-lithography-based system that prints within a given 3D field of
view with simultaneous multi-voxel projection, much like the projection of a hologram (holography) with
voxels (3D pixels) that maintain a print resolution of 0.5 x 0.5 x 3 microns in the x, y, and z dimensions.
The resolution of the system is dependent upon the optical components used to guide and focus the light
source.
Custom optical components can be introduced to decrease resolution or increase the speed of printing.
Subcellular optical resolution (less than about 1 micron), however, may not be required or desired for
printing vasculature embedded tissues. Because the holography process utilizes optics, rather than a print
nozzle for material deposition, the width of the projected laser light can be can be altered on the order of
milliseconds to increase or decrease structural dimensions of the printed materials. Optics-based
holographic printing therefore offers both high resolution and near-immediate changes in resolution that
can be applied to create ultrafine structures.
Printing Speeds Compatible with Organ and Tissue Generation
The polymerization process is controlled by the localized absorption of light, lending flexibility on par with
that of projecting a series of images on a movie screen. Indeed, the holograms utilized to recreate the
structure are projected as a series of images at speeds well over video rate speeds, up to 250 Hz.
Manufacturing of 3D-printed components is dependent upon the speed of layer deposition. In this case,
the layers are three-dimensional and deposited by a light-induced chemical reaction (see Figure 1) that
occurs on the order of 5 or fewer milliseconds. At these speeds, printing time shifts to be more dependent
upon the chemistry of the printing formulation than the physical properties of laser projection. By
removing the mechanical encumbrances of laser light scanning in the case of pinpoint laser printing, or zstep requirements of planar printing, while scaling the manufacturing technique from 1D or 2D, a
significant reduction in print times occurs. This allows biologically complex structures to be created at
unprecedented speeds. Increased print speeds decrease manufacturing times of complex tissues such
that printing of human organs and organ structures is now in the range of possible.
3D Layering Allows for Organ Systems Level Complexity
Beyond meeting requirements of resolution and speed, Prellis Biologics can utilize far-red based light
polymerization to introduce previously undescribed complexities into the 3D printing process. Printing a
fully formed structure inside of another already formed structure is possible using multi-photon
polymerization, where the structure is optically clear to the far-red excitation source. This process is
demonstrated below in Figure 3, where a single sphere is deposited inside of a printed tube.
Figure 3. Demonstration of precision multiphoton printing inside an already formed structure.
To date, the ability to print behind or within another already deposited layer has not been described with
any other 3D printing method. This feature is useful for adding additional tissue layers within or behind
already deposited layers in a multi-layered cell printing process. This capability is critical for building
complex tissues, such as the kidney, especially the glomerulus the primary point of blood filtration. The
glomerulus is comprised of a fine networks of renal vasculature sit within the capsule like a ball and socket
joint, or baseball inside of a baseball glove. Therefore, 3D layering with living cells allows for creation of
structures around finely layered microvasculature and thus the development of fully functional tissues
and in the future, organs.
Biocompatibility of Multi-Photon Holographic Printing
3D printing ink is of paramount importance for building complete tissues. Traditional cell-laden bioinks
can be formulated for multiphoton-based printing through the addition of a photo-initiator. The photoinitiator undergoes a photoreaction on the absorption of far-red (infrared) light, catalyzing the
polymerization of the bioink. There are a variety of bioink/photo-initiator combinations that are
compatible with multiphoton printing, all of which have demonstrated high biocompatibility when
subjected to cell viability studies.
Relative to UV and single wavelength polymerization, far-red multi-photon excitation, provided by a
femtosecond (10 x 10 -15 second) pulsed laser source, ensures that high energy is only absorbed at the
focal plane, therefore the rest of the bioink only experiences brief pulses of far-red low energy light.
Figure 3. A representative graphic of
printing holographically printing a sphere
inside of an already formed threedimensional tube (top row). A hollow tube
structure printed with multiphoton
holography allows for the projection of a
spherical hologram into the center of the
tube depositing the complete sphere inside
of the tube without disrupting the
structure. The sphere is printed in 5 or
fewer milliseconds (bottom row).
Studies using far-red light at various wavelengths have demonstrated far-red laser light to be nononcogenic and protective from oxidative stress in cell culture.75 Accumulated cell damage is pulse-length,
power, and dwell-time dependent; therefore rapid printing and short laser exposure minimizes the risk of
cell death. Furthermore, laser irradiation at low powers has no measurable effects on cell division, at
infrared wavelengths roughly above 1 nm.76, 77
Summary of 3D-Bioprinting Technology
Prellis Biologics is poised to move into the human organ and tissue market rapidly and with a unique
approach that can be scaled to generate whole organs in timeframes that will allow for transplantation in
high-need patient groups. 3D printing approaches that can meet all four requirements of resolution,
speed, complexity and biocompatibility will enable a myriad of functional applications of tissue and organ
engineering. Examples of applications that Prellis Biologics’ technology is uniquely suited for include rapid
single cell encapsulation, organoid printing for disease models, and tissue and organ printing for
transplant.
Prellis Biologics has demonstrated biocompatible single cell encapsulation capabilities at estimated rates
of up to 20,000 cells per second. Using a deterministic, or targeted approach, Prellis Biologics’ holographic
projection of encapsulation spheres is being developed with computer vision capabilities to enable
selection and rapid encapsulation of specific cells based on label or morphology, while preventing
accidental encapsulation of multiple cells at once. Single cell encapsulation enables single cell precision
during the engineering of complex tissues, and will facilitate delivery of cells in vivo for therapeutic uses.
The single cell encapsulation total addressable market is estimated at $1.67B in 2017.
Vascularized organoids for detailed study of human disease models has numerous advantages over
studying cells in a dish and animal model use. The discrepancy between in vitro efficacy and clinical
outcomes can be attributed to limitations of 2D cell culture models. With print resolutions in the submicron range, Prellis Biologics will have the capacity to recreate complex 3D tumor microenvironments
that combine tumor cells with extracellular matrix. This will allow for improved screening of drug
responses and the study of tumor-stroma molecular interactions. The total addressable market for 3D cell
culture systems was estimated at $559M in 2017.
Scalable Human Tissue and Organ Engineering
More than 2 million people die prematurely every year for lack of access to treatment for kidney failure.
92
Numerous chronic illnesses are managed by therapeutic interventions that are both costly with numerous
repeat interventions required and lead to high patient morbidity and mortality. Replacement human
organs and tissues will both help to alleviate sky-rocketing medical costs and allow for patients to regain
functional independence from dialysis, oxygen tanks, multiple daily insulin injections, and other lifealtering therapeutic interventions. Scalable human and organ engineering, using a patient’s own cells to
maximize compatibility is a medical intervention with the true hallmarks of a disruptive technology as it
can be applied to nearly every major disease state. In solving the final hurdle in organ manufacturing, the
ability to rapidly produce a vascular system, holographic 3D printing can support for the first-time largescale tissue manufacturing that will allow for significant advances and pave the way to functional organ
replacements. The size of the human organ and tissue market when estimated by the number of patients
in need, multiplied by the standard procurement cost of a donated organ, totals well over $3T world-wide.
Other approaches to solving the human organ shortage and the impending medical crisis including
recellularization of existing tissue structures and development of animals for xenografts that would allow
for easy transplantation due to removal of immune-system triggering elements. Both technologies offer
promise in specific areas, however both face significant technical hurdles that may be cost and energy
prohibitive in development of full human organ transplantation.
Recellularization of tissue scaffolds involves dissolving cells with detergents, washing the remaining
extracellular matrix, then re-introducing the desired cells by bathing the tissue structure in a cellcontaining media. Recellularization is one approach that has been developed with moderate success in
the laboratory setting. Indeed, visually stunning examples of cellular behavior in decellularized structures
such as cardiomyocyte repopulation of a spinach leaf 78 have inspired the field in the direction of
replacement tissue engineering. To-date one animal transplant with a rat lung lobe has demonstrated
partial success, wherein the lung retained at least partial function for 6 hours but the tissue function
ultimately failed, and the rats required intubation.
79 Furthermore, this process has yet to produce working
blood vessels and microvasculature, however numerous promising studies especially in the area of lung
recellularization are underway. In some skin transplant studies, blood vessels begin to grow into the
transplanted tissues, however thicker portions of tissue rapidly become necrotic due to lack of oxygen.
Another hurdle faced by recellularization is that it does not solve the critical problem of single cell
resolution or placement of cells within finite functional cell niches. For example, airway endothelial cells
and vascular endothelial cells, along with alveolar macrophages must have the correct relative placement
for the lung tissue to differentiate and function properly. In one re-cellularized lung studies, scientists
observed incomplete differentiation of the engrafted cells, noting a lack of mature ciliated or secretory
cells in the conducting airways.
79 Recellularization is effective in painting layers of cells on a tissue scaffold
which works for thin tissues. Ultimately sourcing of the organ scaffold may become a significant
bottleneck that can be alleviated by effective 3D printing of identical cell scaffolds.
Xenograph Transplantation
Xenograft models are particularly attractive source of organs for transplant. Heart valves and ligaments
from porcine, bovine, and ovine sources have been used successfully for decades. These larger structural
elements are chemically treated prior to remove cellular components prior to transplantation to remove
cellular components and there are few reported cases of rejection. Chemical pretreatment is not
compatible with the transplantation of more complex function organs, however, leaving two significant
hurdles preventing the widespread adaptation of xenotransplantation: transmission of zoonotic diseases
and immunological rejection.
Recent advances in gene editing approaches have catalyzed efforts to breed and develop genetically
modified pigs that will produce organs suitable for human transplantation. Much like humans, mammals
carry diseases and viruses that could be transmitted to xenograft recipients. Of concern are porcine
endogenous retroviruses (PERVs), which are inserted into the genome of every pig and can infect human
cells. Recently, scientists have used the genome editing approach, CRISPR, to reportedly eliminate all 62
known PERVS from their genetically modified porcine population.
80 While significant additional work,
particularly in immunological compatibility will be required to optimize this approach in pigs developed
for organ transplant, this represents a positive step in the development of organs for transplantation.
The second significant barrier is rejection due to immune response, or graft v. host disease where the
organ graft brings enough of its own immune cells along to mount an attack against the host. Our immune
systems are highly evolved to reject any foreign material; organs from a porcine source breed stock that
exists today could provoke a significant immune response. Scientists have begun efforts to use gene
editing to modify porcine specific-genes to develop pigs with organs that have improved bio-compatibility.
However, this will be a difficult approach. First, to manipulate the porcine genome to be accepted by the
human immune system numerous genes must be altered. Second, there are thousands of protein
differences between humans and porcine species, and many more are likely to become recognized as
immunogenic. Third, and perhaps the most prohibitive, many of the genes in the immune system and
similar proteins play a significant role in the developmental process of the animal during gestation,
mutations and alterations to these may be a non-starter in developing a breeding population. Finally, even
if most of the factors that cause rejection were eliminated, a patient would likely still require life-long
immunosuppression. Immunosuppression drugs average $20,000 per year and increases the likelihood of
death from aggressive cancer by three-fold across all age groups.
81
An alternative approach would be to grow human organs in host animals using human-pig chimeras. In
recent studies, scientists used gene editing to remove the genes responsible for pancreatic development
in a pig embryo. With the introduction of human stem cells during the gestational period, scientists are
determining if a functional human pancreas will develop inside of the porcine host. While this approach
was successful in mouse-rat chimeras,
82 recent attempts to create human-pig chimeras showed extremely
low rates of human cell engraftment.
83
The xenograft approach is beneficial in that tissue will not be as supply limited as re-cellularization
approaches using human organs and combining the two approaches may prove fruitful for the
development of many tissues for transplantation.
SUMMARY
Fewer than 20,000 organ transplants are carried out each year in the United States. Every year less than
one-sixth of the people on the organ transplant waitlist receive an organ. An estimated 330 people die
every day in the United States due to organ failure. Requirements for being on a transplantation list are
strict; the over 120,000 people waiting are only a fraction of over 90 million people in the United States
who are suffering from some form of progressive organ failure and could benefit from an organ transplant.
Progressive organ failure, specifically kidney failure, is a silent disease that will affect one in seven adults
in the United States, and is a contributor to heart disease,
8, 84, 85 dementia,
86, 87, 88 loss of bone density and
catastrophic fractures,86, 89 and Parkinson’s disease.
90, 91
Advances in immunotherapeutics, vaccines, and treatments for communicable disease have
demonstrated significant impact in health and life-span; however, few advancements have occurred in
the field of organ replacement. As these causes of death are minimized, a new challenge is on the horizon:
how does our health care system cope with the tremendous costs associated with organ failure?
Building human organs with 3D printing not only has tremendous potential for improvement of
therapeutics development but in developing an interventional protocol that will minimize the progression
of the major points of health failure and cost.
Significant issues exist in technological approaches to organ engineering, such as re-cellularization, and
the development of animals for xenotransplantation. Standard 3D printing methods are hindered by the
trade-off between speed and resolution in manufacturing. The slow speed at necessarily high resolutions
does not allow for cell-containing native tissue structures, complete with blood vessels, to be printed fast
enough to keep cells, or a patient, alive.
At Prellis Biologics we have developed a laser-based technology that decouples speed from resolution, in
a biocompatible printing process with capacity to build highly complex structures. Our mission is to
develop this platform technology to build fully functional human organs and tissues on demand. These
organs will represent a disease-free, fully-human, cell-based alternative to donor organ transplantation.
In addition, this technology can create perfectly matched organs that will eliminate the need for
immunosuppression and offer the possibility of removing disease causing genetic mutations. The vision
of Prellis Biologics is to give the millions of patients suffering from progressive organ failure their lives
back.
Acknowledgements: The author would like to thank Richard Hutton, Risa Patterson, Barbara Krause, and
Noelle Mullin, PhD for editing and review of the manuscript content.
Prellis Biologics, Inc. was founded in San Francisco, CA in October of 2016, has filed 2 patent applications
and has raised a total of 3.1M in pre-seed, seed funding from lead investors IndieBio and True Ventures.
For further information please contact info@prellisbio.com
For media inquiries please contact Barbara Krause of Krause-Taylor: barbara@krause-taylor.com
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Artif Cells Nanomed Biotechnol. 2017 Dec 14:1-10. doi: 10.1080/21691401.2017.1416391. [Epub ahead of print]