Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Engineering

Department

Chemical and Materials Engineering

First Advisor

Dr. Bradly Berron

Abstract

There is an urgent demand for engineered tissues and organs because of extremely limited donor tissues available for transplant. The challenge of tissue rejection has motivated many researchers to try to remove all of the cells from a heart and then add back in patient matched cells. These recellularized hearts are a promising avenue towards a functional bioartifical heart that is less likely to be rejected. Decellularized extracellular matrix (ECM) scaffolds generated from animal hearts do not have the cellular components that elicit immune rejection in patient, and they also preserve the native heart tissue structure. While decellularization techniques are sufficiently mature to remove immunogenic species, the current recellularization techniques are unable to distribute and guide the cells to reconstruct the complicated architectures of heart tissue. As a result, none of the hearts made from decellularized tissues have ever exceeded 10% of the pumping capacity of a normal heart. This dissertation develops the techniques to carefully reconstruct the natural cellular organization within heart tissue on a scaffold composed of decellularized heart ECM.

In chapter 2, we develop the fundamental approach for patterning cells onto decellularized ECM. Specifically, patterned light excitation of TFPA group drives the covalent binding of other biological anchoring groups (biotin) only in irradiated regions. THPA reacts with C-H and N-H groups of protein of the tissue, and these regions selectively bind biotinylated cells using a streptavidin bridge. With this methodology, cells can be patterned in arbitrary shapes with size scales from the single cell level (micrometers) up to the tissue level (>cm). We also illustrate the utility of biotin streptavidin interaction to guide the cell orientation after cells are patterned on the substrate. We conclude that on uniform protein substrates, the pattern guides the cell orientation, but on decellularized ECM, the cells orient to the natural desired orientation of the tissue fiber. We report the first time cells have been patterned onto decellularized ECM. This precise patterning of cells at specified locations within a complex tissue has broad application in tissue engineering systems, including vascular, neural, cardiac, and muscle tissue that requires cell alignment.

Even the simplest natural tissues are composed of complex arrangements of multiple cell types. To support increased tissue function, we co-patterned heterogeneous cell types to reconstruct cardiac tissue structures. In chapter 3, we patterned the HUVEC and NIH3T3 cells on the protein coated surface in one step method by antigen antibody interactions, this method showed higher specificity than biotin-streptavidin strategy in previous study. In chapter 4, we studied the mathematical model of single cell attachment on the substrates by the linkages of biotin-streptavidin and antigen-antibody. We first quantified the density of biotin on cell surface and the density of streptavidin on the substrate. Then using the contact area of single cell on the substrate and a microfluidic shearing flow apparatus, we developed a new relationship between the cell-substrate interface characteristics and the adhesive force retaining the cell to the substrate.

The overall goal of this work is to reconstitute the complex tissue that are spatially organized into functionally and morphologically distinct parts that work together to promote tissue function. We spatially and temporally control the position of cells within the tissue to ultimately define cell-cell interactions that drive cellular phenotype and tissue function. In the future, the 2D structures we have studies will be aligned and stacked in complex 3D structures. We expect the ability to rapidly organize multiple cell types with microscale precision into units that combine to generate tissues of scalable sizes will dramatically accelerate the development of artificial tissues.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.408

Funding Information

2019-2020 American Heart Association grant 18IPA34170059

2017-2019 National Heart, Lung and Blood Institute (NHLBI) grant R01 HL127682

2017 and before: National Science Foundation CBET-1351531

Available for download on Saturday, September 24, 2022

Share

COinS