3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Summary

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Abstract

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

Introduction

Traditional cell culture on flattened two-dimensional (2D) surfaces, such as a culture dish or multi-well plates, can hardly elicit cell behaviors close to their native states. Accurate recapitulation of native cellular microenvironments, which comprise of various cell types, extracellular matrices and bioactive soluble factors in three-dimensional (3D) architectures^1,2,3,4, is essential to construct biomimicking tissues in vitro for applications in tissue engineering, regenerative medicine, fundamental biology research and drug dis....

Protocol

Animal experiments followed strict protocol approved by the Animal Ethics Committee on the Center of Biomedical Analysis, Tsinghua University. Under approval of Ethics Committee, human adipose tissue was obtained from Department of Plastic Surgery of Peking Union Hospital with informed consent from the patients.

1. Fabrication of 3D Microcryogels

1. Design and fabrication of microstencil array chips
   1. Use a commercial software to design arrays of specific geometries, such as circles, ellipses, triangles or clovers¹⁴, depending on subsequent application.
      NOTE: Refer to secti....

Results

Fabrication and characterization of microcryogels for 3D microtissue formation.

According to this protocol, microcryogels were fabricated to form the 3D microtissues and individual microcryogels or microcryogel arrays, and were applied to regenerative therapy and drug screening, respectively (Figure 1). Microstencil array chips fabricated from PMMA were applied as micromolds for mic.......

Discussion

Regenerative medicine and in vitro models for drug screening are two important applications for tissue engineering^5,6,7,8,9. While these two applications have vastly different needs, a common ground between them lies in the need for a more biomimetic culturing condition to enhance cell functions¹⁹. Only with improved cell funct.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gelatin	sigma	G7041	All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.
Glutaraldehyde	J&K	902042	Used as crosslinker in preparation of material.
Glass cover slip (24 x 50 mm)	CITOGLASS, China	10212450C	To scrape prcursor solution onto microstencils array chips.
Sodium borohydride, NaBH4	Beijing Chemical Works	116-8	To wash remaining glutaraldehyde away after gelation.
Vacuum jar	asperts, China	VC8130	To preserve microgels under vacuum.
Polymethylmethacrylate (PMMA) sheets	Sunjin Electronics Co., Ltd, China		Ordinary PMMA sheets.
Rayjet laser system	Rayjet, Australia	Rayjet 50 C30	To engrave PMMA sheets to form wells.
Plasma Cleaner	Mycro Technologies, USA	PDC-32G	To make PMMA hyphophilic.
Lyophilizer	Boyikang, China	SC21CL	To lyophilize materials.
Trypan Blue solution (0.4%)	Zhongkekeao, China	DA0065	To dye microgels.
Doxorubicin hydrochloride	ENERGY CHEMICAL, China	A01E0801360010	To test drug resistance of cells in 2D or 3D microgel.
Live/dead assay	Dojindo Molecular Technologies (Kumamoto, Japan)	CS01-10	To distinguish alive and dead cells.
Cell Titer-Blue	Promega (Wisconsin, USA).	G8080	To test cell viability.
Cell strainer	BD Biosciences, USA	352360	To collect microgels.
D-Luciferin	SYNCHEM (Germany)	s039	To tack cells.
Scanning electron microscope	FEI, USA	Quanta 200	To characterize microgel morphology.
Mechanical testing machine	Bose, USA	3230	To measure mechanical features.
Programmable syringe pump	World Precision Instruments, USA	ALADINI 1000	To test injactabiliy.
Digital force gauge	HBO, Yueqing Haibao Instrument Co., Ltd., China	H-50	To test injactabiliy.
Ethylene oxide sterilization system	Anprolene, Anderson Sterilization, Inc., Haw River, NC	AN74i	To sterilize microgels with ethylene oxide gas.
Microplate reader	Molecular Devices,USA	M5	To measure fluorescence intensity in micro-array.
Confocal microscope	Nikon, Japan	A1Rsi	To observe cell distribution in 3D.
Xenogen  Lumina II imaging system	Caliper Life Sciences, USA	IVIS	To track cell in animals.
Liquid work stataion	Apricot design,USA	S-pipette	To load medium or cell suspension high-throuputly.