Optimizing Cell Extraction from 3D Bioengineered Pulmonary Artery Models for Downstream Analysis Restricted; Files Only

Abstract

Efficient recovery of viable cells from 3D bioengineered constructs is critical for downstream molecular analyses, yet dense hydrogel networks pose significant challenges. In this study, we systematically optimized protocols to extract smooth muscle cells (SMCs) from hydrogel-based pulmonary arterial constructs. We first evaluated crosslinking conditions, comparing ultraviolet and blue light and found that blue light produced constructs with lower stiffness, facilitating more efficient cell release. Cell viability within the constructs was confirmed via live-dead staining, indicating preserved cell integrity prior to extraction. Multiple RNA isolation strategies were then assessed, including direct TRIzol treatment and enzymatic digestion using Collagenase II, Dispase II/Accutase, and Hyaluronidase, with additional purification steps such as BSA cushion separation and dead cell removal. While direct TRIzol treatment yielded higher RNA quantities, enzyme digestion-based protocols provided substantially improved RNA integrity, with RNA Integrity Numbers (RIN) consistently exceeding 7, suitable for bulk RNA sequencing. These findings establish a robust workflow for recovering high-quality cells and RNA from 3D hydrogel constructs, providing a foundation for transcriptomic studies of vascular remodeling in pulmonary arterial hypertension.

Table of Contents

Table of Contents

Chapter I: Introduction………………………………………………………………………...01

I.       Pulmonary Arterial Hypertension (PAH) …………………………………………………..01

a)      Pathophysiology of PAH…………………………………………………………….…01

b)     Current Treatment Limitations of PAH………………………………………………..05

c)      Current PAH Models and Limitations…………………………………………………05

II.    3D Bioengineered Models as a Method to Study PAH……………………………………..07

a)      Fabrication Method…………………………………………………………………….07

b)     Use of 3D Models to Study PAH………………………………………………………08

III.  Bioink……………………………………………………………………………………….11

a)      Major Components of Bioink…………………………………………………………..11

b)     Importance of Optimizing Cell Extraction for Functional Assays……………………..13

Chapter II: Methods……………………………………………………………………………14

I.       Bioink Preparation…………………………………………………………………………..14

II.    Cell Culture and Incorporation of Cells into Bioink……………………………………..…16

III.  DLP Printing of Constructs…………………………………………………………………16

IV.  Printing and Preparing Bioreactors …………………………………………………………17

V.     Crosslinking of Casted Constructs………………………………………………………….18

VI.  Stiffness testing………..……………………………………………………………………18

VII.  Live-Dead Assay…………………………………………………………………………...19

VIII.        Perfusion Set Up…………………………………………………………………………19

IX.  Construct Digestion…………………………………………………………………………20

X.     RNA Isolation……………………………………………………………………………….21

XI.  Dead Cell Removal Kit Method…………………………………………………………….21

XII.  BSA Cushion……………………………………………………………………………….22

XIII.        Bioanalyzer……………………………………………………………………………....22

Chapter III. Results…………………………………………………………………………….23

I.       Characterization of 3D Construct Fidelity and Cell Viability………………………………23

II.    Effect of Crosslinking Wavelength on Construct Stiffness and Cell Recovery…………….24

III.  Optimization of Enzymatic Digestion for Cell Isolation from Hydrogel Constructs……….25

IV.  Evaluation of RNA Isolation Methods for 3D Hydrogel Constructs……………………….27

Chapter IV: Conclusion and Discussion………………………………………………………30

References………………………………………………………………………………………………...33

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Engineering, Biomedical
Keyword	Pulmonary Arterial Hypertension 3D Bioengineering
Committee Chair / Thesis Advisor	Dr. Robert Liu, Emory University
Committee Members	Dr. Hananeh Fonoudi, Emory University Dr. Matthew Weinschenk , Emory University

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