Lockwood SBIR Proposal - 1 SBIR PHASE I PROPOSAL COVER SHEET PROPRIETARY
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3.0 Identification and Significance of the Problem or Opportunity:
The supply of donor organs and tissues is severely outpaced by the demand for the same.
Every day, 17 people die from lack of appropriate donated organs [1]. This problem has been
approached through tissue engineering. Many strategies use decellularized animal tissues or
additive manufacturing (3-d printing) in an attempt to create scaffolds on which human cells can
be seeded and grown [2]. Despite limited success, there are severe limitations to both of these
approaches. Decellularized animal tissue presents issues of immunogenicity, high cost,
unsustainability, inaccessibility, unavailability, zoonotic pathogens, and ethicality. Additive
manufacturing presents issues related to the maximum resolution of the technology, affecting
morphology, structure and porosity that prevent essential functions such as vascularization and
cell migration. For these reasons, another approach is necessary.
We see great opportunity in the field of decellularized vegetal (plant and mushroom) tissue for
the use of human tissue engineering. This opportunity is the focus of our proposed set of
experiments. Despite limited attention given to the use of plant-derived scaffolds for tissue
engineering, there have been several examples of success. For example, one team was able to
heal spinal cord injury in a rat model by inserting decellularized asparagus slices into the wound
[3]. Beyond the opportunities presented for tissue engineering, there also exists the opportunity
for using plant scaffolds in drug delivery devices. We suggest that the field of plant-derived cell
scaffolds will present many more opportunities, if given adequate research attention.
4.0 Background, Technical Approach and Anticipated Benefits
4.1 Overall Background Plant-derived material has been used for thousands of years as a
replacement for human tissue. However, it is only recently that plant-derived material has been
used to help native cells regenerate after injury or disease. Plants are an attractive source for
cell scaffolding because they are abundant, inexpensive, non-immunogenic, possess
morphology conducive toward vascularization, pose low threat of carrying zoonotic pathogens,
and are without ethical concerns.
4.2 Related Research Previous research has established that plant-derived scaffolds are nonimmunogenic, and that animal blood vessels can grow through the scaffolding [4]. Fibroblast,
myoblasts, and epithelial cells can be cultured in McIntosh Red apple hypanthium tissue [5].
Plant decellularization methods have been used for hundreds of years, and some are derived
from methods used to decellularize animal tissue [6]. Seeding of human cells into decellularized
spinach leaves has been shown to speed up the infiltration of cells [7], and functioning,
contractile cardiomyocytes have been grown on baby spinach leaves [12]. Arterial input and
venous output were mimicked by grafting together two leaves of the succulent Aptenia cordifolia
species, decellularizing the grafted leaves, then growing human breast cancer cells on it for one
week [8]. This grafting overcame the limitation of plant vasculature's one-way direction.
Although some of these research trials have involved in vivo implantation of the recellularized
plant scaffold, most were merely in vitro. Of the in vivo studies, most were concluded after only
4 weeks, although one spanned 12 weeks. Other researchers have published results in this field
[9-15], there is much work to be done in this field, particularly with regards to creating uniformity
and standardization of protocols.
4.3 Technical Approach Our approach will be to fill in many gaps of knowledge regarding basic
research in the field of plant-derived biomaterials for cell scaffolds in tissue engineering.
Specifically, we will use a mechanized, automated approach to decellularizing leaf, stem, and
root samples from 90 species of plants and 10 species of mushroom. Samples will be run in
triplicate, to capture intraspecies and intraorganismal variation. Four decellularization methods
will be tested on each sample: chemical, detergent-free, enzymatic, and supercritical fluid. Four
recellularization (seeding) methods will be tested on each sample: passive gravitational
seeding, dynamic seeding, rotational system seeding, and vacuum system seeding. Before and
after decellularization, we will test physical (porosity, permeability, volume, morphology,
vascularity), chemical (surface adhesion of cells, degradation rate of cellulose-lignin moiety),
and mechanical properties (Young's Modulus, Ultimate Tensile Strength, Maximum Tangent
Modulus, Elongation at Breakage). After recellularization with two cell types (MSC and
Hematoblast), we will measure cell proliferation, migration, differentiation, and function.
4.4 Innovativeness and Originality of the Proposed Research No previous research has
attempted the type, duration, or scale of research we are proposing here. One innovation we
offer is automation through robotic mechanization, which will allow for collection of much greater
volume and quality of data than ever before. The automation proposed here will lead to scalable
and repeatable experiments. Among many other firsts, to our knowledge we will be the first
research team to attempt to grow Mesenchymal Stem Cells and Hematoblasts in a closed-loop
vascular circuit made from grafting together two leaves of the succulent Aptenia cordifolia
species. It is fortuitous that plant vasculature follows Murray's law in the same way that animal
vasculature does; this fact can place plant-derived scaffolds above 3-d printed scaffolds, which
lack the required spatial resolution to produce good results for allowing vasculature to grow.
Another aspect of our innovativeness is that we will conduct these experiments longer than any
other research team has in the past; most other experimenters ended their studies after 4
weeks, while some went as long as 12 weeks, but ours will last for 50 weeks, which will provide
a more realistic picture of the feasibility of these scaffolds for long-term use within the human
body.
4.5 Anticipated Results and Commercial Applications of Research We anticipate to find that
plant species within the same family will respond similarly to decellularization techniques. We
anticipate that human cells will grow best in plant parts that are thick enough to allow threedimensional movement. We anticipate that human cells will survive beyond the short time
periods previously published by other research teams. We anticipate that human cells will divide
and differentiate sufficiently (given the appropriate growth media with appropriate growth
factors) and this data can be used to eventually produce whole, soft organs such as liver and
kidney. We anticipate that some plant species and plant parts will be more amenable to the
decellularization process than others, and we hope to find patterns that will help predict future
direction of this field with regards to choosing more plant species to investigate further in future
research. Commercial applications are described in section 7.2 below.
5.0 Research Objectives The primary objective of this research is to gather enough technical
data from basic research that complete human organs can be grown on plant-derived scaffolds.
This will allow us to overcome some of these current drawbacks to using plant-derived
scaffolding: First, there is a lack of optimized decellularization protocols. Second, there is limited
characterization of scaffold ultra-structure after decellularization. Third, there is a lack of plant
species-specific decellularization standards. Fourth, it is unclear if biofunctionalization
isnecessary to promote human cell adhesion onto plant scaffolds. Fifth, it is not clear which
species will allow grafting for fluid to flow to and from tissue in a closed loop system required by
mammalian circulation (all plants have a one-way vascular network). Sixth, there is lack of
standardization for seeding cells into the vein network of a decellularized plant scaffold.
Seventh, there is a lack of characterization of mechanical properties of plant tissue, before and
after decellularization. Eighth, it is not clear how best to control scaffold degradation as the
colonizing cells produce their own extracellular matrix. All of these issues can be resolved
through adequate research attention. We will focus on finding the best decellularization and
recellularization methods, as well as recording mechanical properties of plant/mushroom
scaffolds before and after decellularization.
6.0 Research Plan
6.1 Summary of Project Tasks The projects tasks are shown in the following data tables. A
total of 10,800 data points will be collected (10,800 = 4 tables * 100 rows * 27 columns), each
an average of triplicate runs
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7.0 Commercialization Potential
7.1 Description of Company Our company, Cellular Cellulose Solutions, LLC, has the mission
of advancing knowledge to benefit society by eliminating deaths of those who are waiting on
organ donations. This company was founded by R. Nate Lockwood (Principal Investigator),
Edwin M. Land (CFO), and Jay T. Gould (President) in 2021, who all took a class together at
Johns Hopkins University Whiting School of Engineering for Professionals.
7.2 Commercial Applications There are several outlets for commercialization of the
applications we will develop. First, we will license access to our database of information we
generate. This can be licensed to companies who work in the field of regenerative medicine.
Some of these companies include Medtronic, Indee Labs, Pluristem Therapeutics, Baxter
International, Rubius Therapeutics, Mesoblast, BlueRock Therapeutics, and RoosterBio.
Secondly, we will sell Grow Kits to customers. These Grow Kits will have plant seeds,
decellularization liquids, and biopsy punches (or perhaps, frozen human cells, instead), for
adventurous amateur scientists to grow their own cells in plant scaffolds. This can be used as a
home therapy, where people can take control of their own health by growing their own organs
for future use. At a later time, customers can purchase storage space, in our facility, for their
organs. Another goal in commercializing this technology is to take market share from illicit trade
in human organs. Although by nature it is difficult to measure the revenue in an illicit market, it is
estimated that the illicit trade of human organs is about $800 million to $1.7 billion annually [16].
If these illicit organ harvests and implants were done properly under safe conditions, the
estimated transaction costs would be much higher. This presents potential market profit for the
field of regenerative medicine, and our new technology will make this increased profit possible.
7.3 Advantages of Technology over Existing Technologies The advantages of plant- over
animal-derived scaffolds are these: lower cost, more accessible, more sustainable, more
available, more ethical, no risk of zoonotic pathogens, and less immunogenic. Our technology is
better than additive manufacturing efforts such as 3-d printing because plant tissue has a level
of detail in its vascular scaffolding that additive manufacturing cannot match. A secondary
advantage is that this technology will lower the financial barrier imposed by animal and 3-d
printed scaffolds, and this will allow more entrants into the field of tissue engineering. As more
researchers enter the field, more data can be collected, such as best practices for
decellularization, recellularization, and selecting species for growing human cells on plant
scaffolds. The overall effect will be to rapidly accelerate the growth of this new field of plantderived scaffolds.
The advantage of our approach over previous efforts in this field are that we will have a lean,
swift operation that is automated for maximum consistency of experimental results. We will build
the capability to quickly add new plant species and human cell types into the workflow, in order
to rapidly respond to new challenges in regenerative medicine. At the end of the proposed 50
weeks, we will have taken the number of investigated plant/mushroom species from about 40 to
about 140, which represents a significant stride in the progress of the new field of plant-derived
scaffolds
I am a Professor of Materials Science and Tissue Engineering at the University of Oregon, and I
possess the motivation, expertise, leadership, and training necessary to effectively execute this
proposed project.
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B.
Positions, Scientific Appointments, and Honors Positions and Scientific Appointments
2018Ð Present
Professor of Materials Science and Tissue Engineering
University of
Oregon 2016 Ð 2018
Adjunct Professor, Washington University Department of
Engineering 2014 Ð 2016
Columbus City School, Science Educator 2010 Ð 2014
Data Analyst, Celera 2008 Ð 2010
Laboratory Technician, Tissue RegenCorp 2003 Ð
2007
Student, Ohio University Honors 2020
Award for Most Engaging Professor
2019
Excellence in Teaching, University of Oregon 2018
Outstanding New
Professor Award, Washington U. Dept of Engineering C. Contributions to Science 1. My
early publications have focused on the ability of human cells to grow in decellularized plant
scaffolds.
X`9.0 Consultant and Subcontracts Required to Conduct this Research In order to produce the
expected results, we will subcontract Applied Biosciences to help setup the automation
workflow. Because of their reputation from working with J. Craig Venter's Celera Corporation to
sequence several genomes, including the human genome, we have full confidence in their
ability to setup the automated workflow we require. We will contract them to setup our FANUC
robots to automatically decellularize plant scaffolds, and recellularize them with human cells. We
will consult periodically with them regarding issues that will arise in the course of this year-long
set of experiments. We will hire an intellectual property attorney in order to maintain control of
the rights to this technology. This will help us to properly commercialize this technology to its
fullest capability. We will subcontract BioCT Innovation Commons for their biological laboratory
space. We will subcontract Acme IT, Inc. to setup and maintain office phones and computers.
10.0 Equipment, Instruments, Computers and Facilities For this proposed set of experiments,
we will use several companies for physical assets and lab space. We will use HiSpec4 camera
(FastTec, San Diego, CA) on a DMIL inverted microscope to measure cell proliferation. We will
use NIH's ImageJ software to process image files. We will use one FANUC M-10 (mid-grade)
robot for automating the process of placing plant and mushroom parts (leaves, stems, roots,
hyphae) in the decellularization solution; these FANUC robots will also be used to seed human
stem cells onto the decellularized scaffolds. Three Apple iMac computers will procured, to be
used by each of the three members of the core team working on this project. Finally, BioCT
Innovation Commons facilities in Groton, Connecticut will be contracted for renting laboratory
bench space.