BIOLIFE4D Research Presentation APRIL 2022

RESEARCH OPERATIONS
AT BIOLIFE4D
RAVI K. BIRLA, PHD
CHIEF SCIENCE OFFICER

• Introducing BIOLIFE4D
• My credentials to lead R&D at BIOLIFE4D
• BIOLIFE4D Research Labs and Personnel
• Organ Fabrication Process at BIOLIFE4D
• Stem Cell Engineering Technology
• Bioink Development
• Bioprinting Technology
• Bioprinting Cardiac Patches
• Bioprinting Mini-Hearts
• Vascular Grafts
• Bioprinting Heart Valves
• Bioprinting Ventricles
CONTENTS

• Founded in November 2016 to leverage recent advances in
bioprinting technology to bioprint human hearts for clinical
transplantation.
• Business headquarters of BIOLIFE4D are in Chicago, IL and
research labs are based in Houston, TX.
• Steven Morris is the Founder and CEO of BIOLIFE4D and
leads all business development, fundraising and strategic
planning for BIOLIFE4D.
• Dr. Ravi Birla, PhD, is the Chief Science Officer of BIOLIFE4D
and is responsible for all R&D efforts.
• Dr. Jeffrey Morgan, MD, Chief of Cardiothoracic Surgery at
Texas Heart Institute, is Chief Medical Officer at BIOLIFE4D.
INTRODUCING BIOLIFE4D

• BIOLIFE4D is an emerging biotech company focused on
leveraging advances in life sciences, bioengineering,
computational medicine, and additive manufacturing
technologies to 3D bioprint viable human cardiac components.
BIOLIFE4D VISION STATEMENT

• Chronic shortage of donor hearts.
• Number of donor hearts is significantly less than numbers of hearts
required for transplantation.
• Many patients die while waiting for a heart transplant.
• For heart transplant patients
• Immune suppression therapy required
• Qualify of life significantly reduce
• Low long term survival rates
• According to my good friend and CMO at BIOLIFE4D, Dr. Jeff
Morgan:
• By receiving a heart transplants, patients are trading one disease for
another one
• Can we bioprint hearts to solve these problems?
WHY BIOPRINT HEARTS?

MAMMALIAN HEART
Mammalian Heart
Heart Valves
Heart Muscle Heart Chambers
Conduction System Vasculature

DEVELOPMENT PLATFORM
CELLS PLANAR
TISSUE
TUBULAR
GRAFTS
SINGLE
CHAMBER
SOLID
ORGANS
Smooth Muscle Cells
Cardiac Myocytes
Pacemaker Cells
Purkinje Cells
Endothelial Cells
Fibroblasts
Cardiac Patches
Valves
Left Ventricle
Vascular Grafts
Biological Pumps
Mini-Heart

• PhD in Biomedical Engineering from the University of Michigan,
Ann Arbor, one of the premier research institutions globally (2004).
• After my PhD, I was immediately recruited to the University of
Michigan Medical School, Division of Cardiac Surgery, to
spearhead a new research program. I was the Founding Director
of the Artificial Heart Laboratory. (2004-2010).
• In 2010, I moved to Tulane University with a Named Professorship
with the Department of Biomedical Engineering.
• In 2011, I was recruit as the Founding Faculty at the University of
Houston Department of Biomedical Engineering. (2011-2016).
• In 2017, I was recruited to serve as the Associate Director of the
Department of Stem Cell Engineering at Texas Heart Institute.
• In 2018, I moved to BIOLIFE4D to assume the position of CSO.
CSO BACKGROUND

CARIBBEAN ICON
• The Government of Trinidad
and Tobago recognizes local
nationals for outstanding
accomplishments in Science
and Technology.
• Dr. Ravi Birla received the
Caribbean Icon award for
Science and Technology in
2015.
• http://icons.niherst.gov.tt/icon/ra
vi-birla-tt4/
• Dr. Birla has served as a
National Advisor, supporting
the developing of National
Research Labs.

PUBLICATION RECORD
• Dr. Birla is widely
recognized as a thought
leader in the field of
Cardiac Bioengineering
and is well published in
the area.
• 60+ Publications in the
area in top ranked peer-
reviewed journals.
• Dr. Birla’s work has
frequently been featured
on the cover of
prestigious journals.
July 2015
Jan 2015

CREATING AND MANAGING IP
• My first patent was issued in 2003 while in graduate school and I
received my first royalty check as a graduate student.
• I have four issued patents and a fifth one in preparation for filing, all in
the field of cardiac bioengineering, several of which have now lead to
successful commercial products.
2003 Patent # 7,338,798 for Patches 2017 Patent # 9,808,336 for Hearts

Patent Portfolio at BIOLIFE4D
• Methods and Compositions for Organ Printing,, Application
No.: 62/845,162, filed May 8, 2019, United States Provisional
Application BioLife4D Corporation,
• Bioprinting with Cellulose – File in January 2020 – describes a
novel formulation of bioink consisting of cellulose, that is
suitable for applications in cardiac tissue engineering.
Application No.: 62/962,337, filed January 17, 2020

MY BOOKS IN TISSUE ENG
Outlines the principles
of Tissue Engineering
Strategies for
bioengineering the heart

RESEARCH LABS update
• Located at JLABS at TMC, Houston.
• 34,000 square feet incubator
research facility, with 17,000 square
feet of shared space.
JLABS at TMC
BIOLIFE4D Labs

SCIENTIFIC ADVISORY BOARD
Dr. Jeffrey Morgan,
Baylor College of Medicine
Chief Medical Officer
Dr. Adam Fienberg
Carnegie Mellon University
Bioprinting Expert
Dr. Ibrahim Ozbolat,
Penn State University
Bioprinting Expert
Dr. Sean Palecek,
University of Wisconsin
Stem Cell Expert
Dr. Raimond Winslow
John Hopkins University
Computational Modelling
Dr. Shayn Peirce-Cottler
University of Virginia,
Vascularization Expert
Dr. Janet Zoldan
University of Texas
Cell-Material Interface

ORGAN FABRICATION PROCESS
STEP 1
OUR MODEL
STEP 2
STEM CELLS
STEP 3
BIOMATERIAL
STEP 4
BIOINK
STEP 8
TRANSPLANTATION
STEP 6
CONDITIONING
STEP 7
BIOREACTORS
STEP 5
BIOPRINTING

STEPS 1-5 DURING ORGAN FAB
STEP ONE - OUR MODEL: We obtain MRI/CT images of human hearts
that are converted to stl models and the print ready G-code files.
STEP TWO - STEM CELLS: We convert human white blood cells to iPS
cells and then to cardiomyocytes and other cells of the hearts.
STEP THREE - BIOMATERIAL: We use an optimized cocktail of
polymers to formulate our custom biomaterial to bioprint human hearts.
STEP FOUR – BIOINK: We use a custom formulation of cells,
biomaterials and growth factors to formulate our bioink.
STEP FIVE – BIOPRINTING: We use optimized printing parameters to
bioprint hearts and components of the heart like muscle and ventricles.

STEPS 6-10 DURING ORGAN FAB
STEP SIX - CONDITIONING: Growth factors, hormones and
cytokines are used to stability the newly bioprinted heart.
STEP SEVEN - BIOREACTORS: We use custom fabricated
bioreactors for whole heart maturation using media perfusion and
electrical stimulation.
STEP EIGHT – TRANSPLANTATION: Our bioprinted heart is now
ready for patients in need of a heart transplant.

CELLS IN THE HUMAN HEART
HUMAN HEART
FIBROBLASTS
CARDIAC MYOCYTES
PURKINJE CELLS
PACEMAKER CELLS
ENDOTHELIAL CELLS
SMOOTH MUSCLE CELLS
CONDUCTIVITY
CONTRACTILITY
VASCULATURE
STRUCTURAL

SEMINAL PUBLICATIONS
2006: Plasticity of Mature Cells 2009: Conversion of iPS cells to CMs 2012 Nobel Prize

OUR STEM CELL STRATEGY
Donor White
Blood Cells
iPS Cells
FIBROBLASTS
CARDIAC MYOCYTES
PURKINJE CELLS
PACEMAKER CELLS
ENDOTHELIAL CELLS
SMOOTH MUSCLE CELLS

IPS CELLS CYTOGENETIC ANALYSIS
Staining: GTG Sample: ALSTEM/iPS15
Cells: 20 Banding Level: From 400
Karyotype: 46, XY
Conclusion:
Cytogenetic analysis of cell line ALSTEM/iPS15 showed a normal male karyotype by GTG-banding.

IPS CELL CULTURE
DAY 1 DAY 3 DAY 4
DAY 5 DAY 6 DAY 8

iPS Derived Cardiomyocytes
DAY 6
DAY 8
DAY 14

VASCULAR CELLS IN THE HEART
Aortic Endothelial Cells Aortic Smooth Muscle Cells Cardiac Ventricular Fibroblasts

CONDUCTING PURKINJE CELLS
Bright-field MLC2v-GFP Positive Cells Merged Image

CONDUCTING PACEMAKER CELLS
EMA/SHT5

• The objective is to develop custom biomaterial formulations that
mimic the properties of the mammalian heart.
• Soft Biodegradable Hydrogels:
• Collagen, gelatin, fibrin, agarose, alginate.
• Criteria for biomaterial development:
• Printability – extrusion temperature, speed and pressure.
• Mechanical Properties – Young’s modulus.
• Degradation kinetics – time dependent change in material mass.
• Biocompatibility – responsiveness to cell growth.
BIOMATERIAL DEVELOPMENT

BIOMATERIAL DEVELOPMENT
• Optimization of biomaterial formulation using: alginate, gelatin, fibrin,
agarose and pluronic acid.
Alginate: 1% to 10% Gelatin: 1% to 10%

Print File: No Fill
Print Pressure: 48-50 kPa
Print File: Infill
Print File: Patch
PRINTABILITY OF BIOMATERIAL
• Example: 10% alginate solution.

BIOMATERIAL 1: FORMULATION
• Alginate concentration of 6-10%, gelatin concentration of 6-10%,
fibrinogen concentration of 0.5-5%.
• Biomaterial 1 Formulation:
• Material Based Applications: 6% alginate, 5% gelatin, 0.5% fibrinogen.
• Cell Based Applications: 6% alginate, 8% gelatin, 0.5% fibrinogen
• Crosslinking Formulation 1: 100 mM calcium chloride, 10 units/ml
thrombin.

SOFTWARE GUIDED BIOPRINTING
STL File Created in Autodesk 360
G-CODE File Created in Repetier
STL File Modified in MeshMixer

EXTRUSION BASED BIOPRINTING
• Dual printhead extrusion based system
• Pressure range 10-300 KPa
• Temperature range from 20oC to 130oC.

BIOPRINTED CARDIAC PATCHES
STL Model
15 mm
16
mm
Bioprinted Cardiac Patch
Bioreactor Hardware Bioreactor Software Bioreactor Video

MINIHEART TESTING APPLICATION
• Goal is to bioengineer mini-hearts that can be used for cardiotoxicity testing.
• Mini-hearts are bioengineered, scaled down human heart models which
provide the necessary functionality required to satisfy testing requirements.
• Scaled down version of a human heart will provide better predictive results of
how a human heart will react as compared to a model involving a different
species.
• Currently, cardiotoxicity testing is conducted using small animal models which
are inaccurate and often provide misleading results.
• Huge market demand - Billions of dollars are wasted each year on human trials
which fail after being introduced utilizing inaccurate animal model testing.

• Criteria established concurrently with CRO
• End point metrics - 1) measurable LV function and 2) measurable
electrophysiology (which ultimately results in the wave of contraction).
• Once real measurable function is via the intraventricular pressure
measuring devices (preload balloon and LV catheter), we will generate a
number of things related to behavior of contractility and ECG
• bowditch effect, anrep effect, frank-starling curves, pressure-volume relationships,
pacing protocols, demonstrable lead II ecg with intervals and morphology (PR, QRS,
QT)
• Evaluate cell maturity, sacromeric structure, contraction and ion channel
expression, t-tubule structure, force frequency relationship.
MINI-HEARTS – Initial (Pre-CRO) Testing

MINI-HEARTS – Initial (Pre-CRO) Testing
1. STRUCTURAL Status 4. FUNCTIONAL Status
Printing heart structure Yes LV Pressure of 120 +/-
10% mmHg
(*)
Internal structures, atrium, ventricle,
valves, etc.
Yes Electrical Conduction
Velocity of 0.5 +/- 10%
m/s
(*)
Necessary cell Types Yes Pressure Volume Loops (*)
Vascularization (TBD what level at all
if required )
Yes (when printed
independently)
Pacing Characteristics
7 Hz for 4 Hours
(*)
2. STABILITY OF PRINTED STRUCTURE Status Minimum Viability Time
(4 days)
Yes (based on individual constructs);
TBD for beating heart
Ability to maintain in culture for 2
weeks minimum
Yes (*) The mini-hearts thus far have not
exhibited consistent, prolonged beating
and therefore these tests can not yet be
completed
3. PRINTING WITH CELLS Status 5. HISTOLOGY (Maturity) Status
Sterility of Printed structures Yes Sacromeric Reticulum Verified post “Functional Testing”
Cell viability in culture Yes T-Tubule Structure Verified post “Functional Testing”

MINI-HEARTS – CRO Testing
• Group 1 - Compounds which have already undergone testing
(so results are known) that showed no sign of negative
cardiotoxicity effects with animal model and ended up being
safe in human trials.
• Group 2 - Compounds which have already undergone testing
(so results are known) that showed no sign of negative
cardiotoxicity effects with animal model but ended up having
negative cardiotoxicity effects on humans.
• Testing is conducted by CRO in GMP lab with 2 dozen
compounds from each group.
• Estimated time to complete tests is 30 days.

BIOPRINTED AORTIC VALVE
STL MODEL

BIOPRINTED LEFT VENTRICLE
Human Neonate
STL Model

SUMMARY
• We have an outstanding team of Scientists working with us.
• We have made great strides in our research endeavors.
• Stem cell engineering
• Biomaterial Development
• Bioprinting
• Bioprinting tissue
• We are well positioned with our stem cell engineering studies.
• We have proof of concept studies on cardiac patches, ventricles,
aortic valves and vascular grafts.
• We are well positioned to build on this work and continue our
journey to bioprint human hearts.
• Our immediate short-term goal is to scale up our research
operations to achieve these targets and develop a mini-heart ready
for cardio-toxicology testing.

BIOLIFE4D Research Presentation APRIL 2022

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BIOLIFE4D Research Presentation APRIL 2022