Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Summary

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Abstract

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.

Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues.

Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

Introduction

In recent years, a variety of technologies have become available that addresses the need for alternative sources of functional organs and tissues by seeking to manufacture, or biofabricate, them. Bioprinting has emerged as one of the most promising of these technologies. Bioprinting can be thought of as a form of robotic additive fabrication of biological parts, that can be used to build or pattern viable organ-like or tissue-like structures in 3 dimensions.¹ In most cases, bioprinting employs a 3-dimensional (3-D) printing device that is directed by a computer to deposit cells and biomaterials into precise positions, thereby recapitulating anatomically-mim....

Protocol

1. Hydrogel Bioink Formulations and Preparation

1. In order to provide tissue-specific biochemical profiles, prepare tissue-specific ECM digest solutions as previously described for liver.²⁰
   Note: In general, this ECM digest will comprise 40% of the final hydrogel bioink volume that is employed. Several hundred milliliters of ECM digest solution can be prepared, aliquoted, and frozen at -80 °C for future use.
2. Prior to hydrogel formulation, dissolve a photoinitiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, in water at 0.1% w/v.
   Note: Volumes in the 50-100 ml range can be prepared ahead of time and sto....

Results

When the procedures described above are followed correctly, hydrogels should contain a biochemical profile specific to the target tissue type,²⁰ allow for a high degree of control over bioprinting and final elastic modulus,³⁴ and support viable functional cells in tissue constructs.

Hydrogel Customization
To best mimic native liver, the hydrogel bioink was supplemented by liver ECM solu.......

Discussion

There are several components that are critical to consider when attempting to biofabricate 3-D tissue constructs, for eventual use in humans or for in vitro screening applications. Employing the appropriate cellular components determines the end potential functionality, while the biofabrication device itself determines the general methodology for reaching the end construct. The third component, the biomaterial, is equally important, as it serves dual roles. Specifically, the biomaterial component must be compati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hyaluronic acid	Sigma	53747
Gelatin	Sigma	G6144
2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone	Sigma	410896
Hyaluronic acid and gelatin hydrogel kit (HyStem-HP)	ESI-BIO	GS315	Kit contains the components Heprasil (thiolated and heparinized hyaluronic acid), Gelin-S (thiolated gelatin), and Extralink (PEGDA)
PEG 8-Arm Alkyne, 10 kDa	Creative PEGWorks	PSB-887
Primary human hepatocytes	Triangle Research Labs	HUCPM6
Primary human liver stellate cells	ScienCell	5300
Primary human Kupffer cells	Life Technologies	HUKCCS
Hepatocyte Basal Media (HBM)	Lonza	CC-3199
Hepatocyte Media Supplement Kit	Lonza	CC-3198	HCM SingleQuot Kits (contains ascorbic acid, 0.5 ml; bovine serum albumin [fatty acid free], 10 ml; gentamicin sulfate/amphotericin B, 0.5 ml; hydrocortisone 21-hemisuccinate, 0.5 ml; insulin, 0.5 ml; human recombinant epidermal growth factor, 0.5 ml; transferring, 0.5 ml)
Triton X-100	Sigma	T9284	Other manufacturers are ok.
Ammonium hydroxide	Fischer Scientific	A669	Other manufacturers are ok.
Fresh porcine cadaver tissue	n/a	n/a
Lyophilizer	any	n/a
Freezer mill	any	n/a
Bioprinter	n/a	n/a	The bioprinter described herein was custom built in-house. In general, other devices are adequate provided they support computer controlled extrusion-based printing of hydrogel materials.
Hanging drop cell culture plate	InSphero	CS-06-001	InSphero GravityPlus 3D Culture Platform