Developing a Micro-Tissue-Engineered Neural Network Using a Hydrogel-Based Micro-column

Abstract

Source: Struzyna, L. A. et. al., Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling. J. Vis. Exp. (2017)

This video demonstrates the development of micro-tissue-engineered neural networks using a hydrogel micro-column with an extracellular matrix core. Seeded neuronal aggregates adhere, extend projections, and form structured neural networks, contributing to advancements in neurodevelopment and neurotherapeutic research.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Development of the Agarose Hydrogel (Acellular Component of micro-tissue engineered neural networks (micro-TENNs))

1. Agarose solution preparation
   1. Within a biosafety cabinet, prepare reservoirs for the micro-columns by transferring 20 mL of Dulbecco's phosphate-buffered saline (DPBS) to each of two 10-cm Petri dishes. Sterilize fine forceps and microscalpels with a hot bead sterilizer.
   2. Weigh 3 g of agarose and transfer it to a sterile beaker inside the biosafety cab....

Results

Figure 1: Blueprints of the laser-cut micro-column fabrication device and the 3D-printed pyramidal micro-well mold.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Laser cutter	Universal laser systems	PLS4.75	Used to fabricate the laser-cut micro-channel mold.
Laser-cut micro-column fabrication device	Custom-made	--------------	Contact our research group if interested. Dimensions and blueprints provided in the manuscript.
Screws	--------------	--------------	#4-40 with a thread diameter of 3.05 mm
Nuts	--------------	--------------	#4-40 with a thread diameter of 3.05 mm
Acupuncture needle (180 µm diameter)	Lhasa medical	sj.16X40	The diameter may be varied according to the desired size for the micro-column lumen.
Petri dish	Fisher	08772B
Dulbecco's phosphate buffered saline (DPBS)	Invitrogen	14200075
Polystyrene disposable serological pipet	Fisher	13-678-11D
Agarose	Sigma	A9539-50G
Capillary tube (398 µm diameter)	Fisher	21170D	The diameter may be varied according to the desired size for the micro-column shell.
Hot plate	Fisher	SP88857200
Magnetic bar	Fisher	1451352
Micropipette	Sigma	Z683884-1EA
25 mm gauge needle	Fisher	14-826-49
Microscalpel	Roboz surgical	RS-6270
Scissors	Fine science tools	14081-09
Forceps	World precision instruments	501985
Hot bead sterilizer	Sigma	Z378550-1EA
Stereoscope	Nikon	SMZ800N	Used for all dissection steps and for micro-TENN fabrication.
Rat tail type I collagen	Corning	354236	Maintain at 4 ºC and remove only when needed. Use ice to preserve its temperature when in use.
Microcentrifuge tube	Fisher	02-681-256
Mouse laminin	Corning	354232	Maintain at 4 ºC and remove only when needed. Use ice to preserve its temperature when in use.
Neurobasal medium	Invitrogen	21103049	Basal medium for the culture of pre-natal and embryonic neuronal cells. Store at 4ºC and warm at 37 ºC before use.
Sodium hydroxide (NaOH)	Fisher	SS2661
Hydrochloric acid (HCl)	Fisher	SA48-1
Litmus paper	Fisher	09-876-18
Hank's balanced salt solution (HBSS)	Invitrogen	14170112	Store at 4 ºC.
0.25% Trypsin-EDTA	Invitrogen	25200056	Store at -20 ºC and warm at 37 ºC before use.
Bovine pancreatic deoxyribonuclease (DNase) I	Sigma	10104159001	Store at -20 ºC and warm at 37 ºC before use.
B-27 Supplement	Invitrogen	12587010	Supplement added to neurobasal medium for the culture of hippocampal and cortical neurons. Store at -20 ºC and warm at 37 ºC before use.
L-glutamine	Invitrogen	35050061	Store at -20 ºC and warm at 37 ºC before use.
Sprague Dawley embryonic day 18 rats	Charles River	Strain 001
Pasteur pipette	Fisher	22-042816
15 mL centrifuge tube	EMESCO	1194-352099
Vortex	Fisher	02-215-414
Centrifuge	Fisher	05-413-115
Hemocytometer	Fisher	02-671-6
Objet30 3D-Printer	Stratasys	--------------	Used to fabricate the pyramidal micro-well molds.
3D-printed pyramidal well mold	Custom-made	--------------	Contact our research group if interested. Dimensions and blueprints provided in the manuscript.
Polydimethylsiloxane (PDMS) and curing agent	Fisher	NC9285739	Comes as kit with elastomer and curing agent. Use inside a chemical fume hood.
Funnel	Fisher	10-348C
1 ml pipette bulb	Sigma	Z509035
Micro-spatula	Fisher	S50821
12-well culture plate	EMESCO	1194-353043
Oven	Fisher	11-475-154
Incubator	Fisher	13 998 076
Eclipse Ti-S Microscope	Nikon	--------------	Used for taking the phase-contrast images. With digital image acquisition using a QiClick camera interfaced with Nikon elements basic research software (4.10.01).