Bioprinting 101: Learn How to 3D Bioprint

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Bioprinting is the application of 3D printing to the field of tissue engineering. On a practical level, bioprinting is the best way to perform cell culturing in three dimensions. It allows one to have microscopic control over cellular environment variables and macroscopic control of geometry.  This is important because the cells in the human body function in a fully three-dimensional environment. Recreating this environment gives researchers the ability to work with more physiologically relevant models of living tissue. Read this bioprinting 101 guide to learn how to 3D bioprint in your lab.

This article outlines the basic knowledge that experienced tissue engineers use to run experiments and inform their further research. The order of topics below follows the natural flow of a bioprinting experiment process. Bioprinting involves the selection and preparation of biomaterials, as well as cell culture for biostudies. Material characterization is an important step to confirm that the construct will have your desired properties. The bioprinting itself requires experimental planning and setup, and it often takes place in a biosafety cabinet. After completing a biostudy, analysis confirms the viability and construct properties. We walk you through all of these steps below and include links to helpful pages on this website for more details on each topic so you can learn how to 3D bioprint. Let’s get started!

Learn to 3D Bioprint Section #1: Bioprinting Preparation

Selecting a Cell Type

The type of cells or cell line you would like to work with will determine many of the constraints for the rest of your printing process. Some tissues, like connective tissues, are fairly well understood and can be grown into robust structures with relative ease. Other tissues, like nerve tissues, can be difficult to work with. It is important to strike a balance between clinical relevance and ease of use for your cell line. You may also need to consider the maturity and source of your cells. Some mammalian, non-human cells like porcine or equine derived samples are popular as are many different stem cells. Your choice of cell might also be determined by the ink you would like to work with as different inks are suited to different cell lines. Below, we begin the discussion of different bioinks and biomaterials.

Overview of Bioink Types

A bioink is a biomaterial that can be used in 3D bioprinting, an emerging method of combining additive manufacturing with 3D cell culture. The material properties greatly affect a bioink’s interactions with cells and its success as a bioprinted construct. One way to make sense of the wide range of materials used in bioprinting is to sort them by their roles in the printing process. A given bioink can be a matrix ink, a support ink, or a sacrificial ink.

Matrix Inks

Matrix bioinks hold and support cells, an important role in bioprinting. Most matrix bioinks are hydrogels, meaning they are primarily composed of water. Bioinks currently available on the market  offer strengths and weaknesses, often meeting some but not all requirements necessary for bioprinting. Reagents in other categories can help compensate for these weaknesses. See the table below for some examples of matrix inks and their main uses. Click here to learn more.

Reagent Application Source Resolution (mm) Sacrificial Support? Curing Reagent
AlginateSoft TissueNatural0.15 +/- 0.03FRESHCalcium Chloride (CaCl2)
CollagenSoft TissueNatural0.32 +/- 0.03FRESHNone
Gelatin Methacrylate (GelMA)Soft Tissue, Hard TissueNatural0.40 +/- 0.03NoneLAP 
PhotoHASoft TissueNatural0.50 +/- 0.03NoneLAP 

Support Inks

Support bioinks are biocompatible materials that provide better mechanical properties than matrix inks. It is common to use support inks to bolster bioprinted constructs since hydrogels tend to be soft with low structural integrity. Support bioinks are also often used for connective or hard tissue applications, such as the fabrication of cartilage, bone or muscle tissues. See the table below for some examples of support inks and their use conditions.

Bioink Melt Temperature (°C) Resolution (mm)
Hyperelastic BoneRoom Temp0.30 – 0.40
Polycaprolactone (PCL)600.16 +/- 0.04
Poly (lactic-co-glycolic acid)(PLGA)72-770.15 +/- 0.01

Sacrificial Inks

As their name implies, sacrificial bioinks are designed to be deposited as temporary support and then removed in post-processing. They are usually used for the creation of complex negative geometries such as vascular networks. In some cases, they can also be implemented to improve the resolution of prints made with materials that have poor shape fidelity on their own. See the table below for some examples of support inks and their use conditions.

Reagent Method for Removal Matrix Bioink Compatibility
Pluronic F127CoolGelMA, Collagen, Alginate
Carbohydrate GlassHeatPDMS (Organ-on-a-chip)

Other Biomaterials

Bioprinting relies heavily on using biocompatible materials in order to create microenvironments that mimic biological systems.

Support Baths

While support and sacrificial bioinks can offer permanent or temporary reinforcement, this does not help constructs that are solely comprised of soft hydrogels or otherwise too geometrically complex. Methods such as FRESH involve printing into a support bath, enabling the creation of such structures.

learn how to 3d bioprint - support baths
Proximal tubule STL file (left), bioprinted tubule in FRESH slurry (center), post-processed tubule out of FRESH (right)

Additives

By encapsulating cells in bioinks, the cells are able to grow and remodel the bioprinted construct in a living tissue. Being in a native bioink helps them do this because it has native proteins. Proteins are unique to different tissue types and add specificity to bioinks, allowing them to be customized to a certain application. Proteins such as vitronectin or fibronectin are used in the body to regulate cell behaviors such as adhesion, migration, proliferation, and differentiation and mediate interactions with collagen, heparin, and GAGs.

Photoinitiators

Photocrosslinking is one of several crosslinking options that increase the hardness of a matrix ink which can be desirable in some constructs. Photocrosslinking in particular uses the energy in electromagnetic radiation to activate the hardening process. Photoinitiators are substances that introduce the necessary free radicals to a matrix bioink to allow photocrosslinking. They vary primarily in the range of the electromagnetic spectrum that can activate them.

Guide to 3D Models

Before you can 3D print something, you have to know what that object looks like. Whether that object is a simple test cylinder or a complex blood vessel network, you usually start with a 3D model, convert that model to an STL file, then convert that STL file to a gcode file which your printer interprets into motor movements. Below, we discuss how to take your print object from idea to STL.

Design it Yourself

There are many programs that will let you design a 3D model yourself. These are often referred to as CAD (Computer Aided Design) programs. SolidWorks, Inventor, and AutoCAD are all popular options, but there are plenty that you can explore. The main advantage of designing your own model is the control you have over the shape. Once you have sculpted your model to your preferred specifications, you can save your CAD file as an STL file and then you are ready to continue to slicing.

If you are a Pro Software subscriber you will also have access to the Integrated Shape Editor which lets you originate and manipulate cylinders, prisms, and droplets all in the same user interface as the rest of your printing.

Convert from Medical Imaging

Another technique many researchers use when developing their 3D models is to convert medical imaging samples to STL files. CT scans are the most commonly converted type of imaging but most forms can be used, especially if they can be saved as a DICOM (Digital Imaging and Communications in Medicine) file. The conversion is performed by software which translates the light intensity recorded in the imaging sample to a corresponding tissue type, and determines the bounds between tissues. Programs such as Mimics, 3D Slicer, and InVesalius 3 are all popular options. The main advantage of converting medical imaging results is the clinical relevance of the models produced.

Use a Database

If you don’t have access to clinical imaging samples, there are many databases with DICOM derived STLs to choose from. Many academic institutions have their own libraries and private collections exist that offer licensing agreements. You can find a list of databases below.

Print parameters

Each material used in 3D bioprinting requires different settings to get optimal extrusion and clean lines. Print parameters can affect outcomes on a microscopic level, such as cell viability, as well as macroscopic outcomes, such as pore size and structural fidelity. Below we discuss the key parameters for the pneumatic extrusion bioprinting process with Allevi bioprinters.

Slicing parameters

Slicing, or the process of converting an STL into a gcode file, requires different settings that tell the software how to partition the STL into layers and the printer how to move to create the construct. These settings include layer height, nozzle size, and print speed. In general, layer height is closely related to nozzle size since nozzles tend to have circular cross sections. It’s important to note that print speed refers to the motion of the extruder relative to the print surface, not the flow rate of the extruded material. Variations in material properties such as viscosity cause different materials to perform better with different settings. For example, some materials are very viscous and extrude slowly, requiring a slow print speed to match.

It is important to note that the limiting factor for printed biomaterial resolution is the diameter of the needle. Resolution also generally improves with lower layer heights and higher print speeds. However, extrusion becomes easier with larger nozzle sizes, larger layer heights, and slower print speeds. The balance between these parameters is often the subject of print optimization which is discussed below. After choosing these parameters, you can slice an STL into a gcode.

Infill refers to the material inside the characteristic surfaces, or shells, of your print. In other 3D printing applications infill is usually defined by geometry and % volume occupied. The Allevi system gives you more precise control of your final construct by letting you set the distance between the infill lines in your chosen infill pattern. The shape and amount of infill you need will vary with the purpose of your construct and the cell line you are using.

Printer Parameters

Once your gcode is ready, you still need to enter the correct printer settings for your material. In the Allevi pneumatic extrusion platforms these include pressure, temperature, and crosslinking.

Over time, we’ve optimized parameters that work well for some common biomaterials. These general print parameters are great for just getting started with a new material and as a starting point for further optimization.

Needle

Needle features such as gauge, length, profile, and material significantly affect the pressures, temperature,and speeds you can use. While it’s typically obvious what the best choice for your needle is, use our guide to picking your needle to weigh all the factors.

learn how to 3d bioprint

Crosslinking

Crosslinking is a process by which the molecules of a soft network change their interactions, causing the material to stiffen. This can be achieved by several mechanisms discussed further below. Allevi printers  incorporate LED’s that can be used in photocrosslinking. The Allevi 3 platform also incorporates a heated bedplate for thermal crosslinking. Chemical crosslinking can also be utilized with the help of printer accessories like petri dishes and coaxial printing nozzles.

Optimization

Applications vary and so do your target specifications for your end construct. Maybe you’re going for speed and high throughput, or maybe you’re more focused on fine resolution. Either way, optimizing print parameters is a key step.

Build plates

Common types of buildplate 

There are three basic types of build surfaces: glass slides, petridishes, and wellplates. Slides are useful for moving a construct over to a microscope for imaging. Petridishes offer a very simple build environment suited to large constructs and testing. Wellplates offer convenient sorting for assays. The Allevi platforms are designed to work with industry standard pieces in each of these types. In fact, the autocalibration function in the Allevi 1 and Allevi 3 platforms is programmed to compensate for the manufacturer specified depths of support build surfaces, ensuring a precise calibration and printing experience.

Bowing and curvature 

Some build surfaces exhibit bowing, or a convex arch in their base. This can create a problem for extrusion bioprinting since the material deposition occurs in layers. If one point of the build surface is higher than another, you run the risk of scraping against your build surface with a low pass or imprecise deposition with a high pass.

Glass vs plastic

While most petri dishes and wellplates are plastic, glass is an alternative that can have benefits.Glass has a higher temperature tolerance and is chemically resistant while some plastics may react to organic solvents used in some bioinks.  Glass also experiences less bowing. However, given the same dimensions, glass is heavier and more expensive. Additionally, broken glass can pose a hazard. You can put cell friendly coatings on either. For some printing with viscous substances, both might be too smooth for precise deposition. In this case, you can use different print surfaces to increase the friction of your surface.

Substrate stiffness

Cells survive best in biomimetic environments. As such, the geometry and stress profile of a cell’s environment can influence cell viability and differentiation. To get the best results out of your cell it may help to introduce an additional structure to your build surface to alter the conditions of a plain slide, plate, or dish.

Heating/cooling

The mammalian cells used most frequently in tissue engineering benefit from being kept in a temperature range near mammalian body heat. To help reproduce this effect, the Allevi 3 system has a heated bedplate that can be raised to temperatures as high as 60°C. This can be especially useful for longer prints.

Some materials are very temperature sensitive and the mechanical properties of a construct may be affected by the temperature maintained in the build environment. For some materials, like collagen, it might help to perform a print in a temperature controlled (cold) room. For other materials, the different temperature of a build surface might be enough to cause thermal crosslinking, adding structural integrity to a construct.

Learn to 3D Bioprint Section #2: Cell Culture

Cell culture remains an important aspect of 3D cell culture and 3D bioprinting. This is due to the large amounts of cells that are required for bioprinting studies. For cell-laden bioinks, concentrations can range from 1-10 million cells per milliliter of bioink. A single bioprinting experiment can easily require 50 million cells.

You can find cell culture protocols on our Protocols page. The Allevi Shop makes it easy pair your bioinks with your cell lines and cell culture reagents.

Learn to 3D Bioprint Section #3: Material Characterization

After choosing an ink to try and preparing a file, it is often important to verify that your ink choice and shape will work for your application. Below we discuss some material characterization steps that are often performed before doing a full bioprinting study. Tests performed are often based on your research goals.

Rheology

Rheology is the study of flow. It seeks to characterize the fluid behavior of substances. In bioprinting, we are primarily concerned with the viscoelasticity of our bioinks. Generally, you want a bioink to exhibit shear thinning, the property of behaving more like a liquid in high shear stress and more like a solid in low shear stress. This is because the material will go through high shear stress as it is extruded (making it more like a liquid and easier to extrude) and low shear stress on the buildplate (making it more like a solid and more suitable for forming a construct).

Other processes like FRESH method printing also use the shear-thinning behavior of a support slurry to allow multi-directional printing without support structures. 

Solid Mechanical Testing

Researchers often want to gauge the mechanical properties of their constructs after they finish printing. The nature of the test should gauge the response of the construct to biomimetic forces. This varies widely based on the design and intended use of the construct. Constructs that need to handle pulling forces, such as ligament or tendon grafts, are often tested primarily for their tensile properties. Constructs that have to bear weight or repeated impact, such as meniscus or bone grafts, are often tested for compressive properties and cyclic loading properties. ASTM and ISO standards often exist for comparison of a novel material to a tested artificial or existing biological sample.

Biocompatibility Tests

If a novel material or construct is being used, it is uncertain how cells will respond to the new conditions created. As such, a key step in the research is proving that cells can survive and proliferate in that material or construct. Typically, a live/dead test can be used to determine the percent viability of cells, while a prolonged metabolic assay can measure the degree of proliferation. This is a good indication not only that cells can survive but that they are able to get the necessary resources to reproduce. Additional tests are often performed to gauge function. One common example is tagging different kinds of cells to gauge the differentiation of stem cells.

Experimental Design

Day-of Preparation

Our guide to biostudy preparation will show you want to do the day of your biostudy.

When working with living cells in a bioink, it is important to maintain a sterile printing environment. It is also important to make sure that your cells mix well into your bioink. Check out our graphic for cell-mixing.

Printing

With your print file optimized and your material ready in a syringe, it’s time to involve the bioprinter. For a reminder on how to control your Allevi bioprinter, visit our getting started page.

For detailed instructions on printing with certain materials, check out our Protocols page.

Post processing

After your print is finished, you may need to do some post processing. This might mean photocrosslinking, removing sacrificial inks, seeding with cells, or any number of other modifications. Additionally, for constructs that include cells, you will need to perform further cell culturing, likely in an incubator.

Analysis

A bioprinting experiment is only as helpful as the conclusions you can draw from it. To get meaningful data from a bioprinting experiment you need to do some analysis on your cultured construct. Fortunately, there are many standard tests and lots of similarities to 2D cell culture that can be an excellent starting point.

Assays

Assays are tests performed over groups of cells to determine trends within a tissue. They are usually used to gauge things like cell viability or functionalization. Assays can use different approaches to inform on the same metric, such as viability, which allows flexibility in your experiments. A key difference in 2D and 3D assays is the importance of assay penetration. In 2D cultures, all cells are easily exposed to the chemical. However, in 3D cultures, cell exposure depends on how far into the construct the assay is able to penetrate. Some assays are sacrificial, meaning that you must first add an enzyme to break up the construct before you can run the assay. Afterwards, you cannot run anymore tests on that construct. However, other assays can better penetrate constructs and allow you to run prolonged tests on the same construct.

Imaging

Imaging on its own produces qualitative data. Incorporating software like ImageJ can allow for pixel-by-pixel data collection to derive more quantitative results. Histology is the practice of taking a cross-section of tissue and staining them to observe how tissue is layered. This is commonly used in in-vivo studies to show wound healing and construct incorporation after the insertion of a print.

Live/Dead imaging is common in 2D cell culture and is also useful for analyzing your bioprinted construct. For detailed instructions, check out our protocols on 2D Live/Dead quantification using Fiji and 3D Live/Dead quantification using Imaris.

Mechanical Testing

Once you have a cultured bioprint available, you should repeat tests from the material characterization phase to determine how cell proliferation and integration has affected your construct.

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