Introduction: 3D Printed Scaffolds for Cell Culture
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Art x Technology x Biology
I am an Artist who uses the body, biology and technology to create artwork. www.amykarle.com
As Artist in Residence at Autodesk, I embarked on creating artwork using the building blocks of life: live cells.
I turned to synthetic biology and regenerative medicine and set out on a journey of creating artwork that could grow into form. Using CAD design and 3D printing, I created scaffolds to encourage cell growth into a certain form, a 3D printed framework that tissue can regenerate on.
There has been a lot of curiosity from the bio-medical, pharmaceutical and cosmeceutical industry and a wide spectrum of people to understand how I created the 3D printed scaffolds. I have been asked on multiple occasions if I would share details on the process.
In this Instructable I share how I create 3D printed lattices (scaffolds) for cell culture. I've also included a sample file for you to try on your own.
This process can not only be used for cell culture but could potentially be applied to design and engineer custom bone grafts or other tissue for medical implant. See whitepapers linked in the resources section of this article for further information.
Alternatively, this software and part of this workflow could also be utilized for the design of metal medical implants. Refer to Within Medical for more details.
Please leave suggestions for other potential applications of this workflow/knowledge in the comment section below!
This project was made possible with the generous support of Autodesk's Pier 9 Artist in Residence Program, Bio/Nano Research Team, the Ember 3D printer team and their material scientists, Autodesk software and software evangelists, The Exploratorium and the California Academy of Sciences.
A very special thank you for all of the support and to all of the special people involved with this project!
Step 1: Ideal Scaffold Characteristics
Ideal Scaffold Characteristics / Objectives:
- Highly porous interconnected network that allows for cell growth and movement of nutrients and metabolic waste
- Non-toxic and biocompatable
- Encourages cell attachment and proliferation
Added requirements for my project:
- Structural properties to encourage stem cell transformation into bone cells
- Biodegradeable with controlled degradation so that the scaffold can be replaced by bone cell mineralization
Step 2: Process Overview
- Optional: Import CT or 3D Scan Data then process using ReMake
- Create initial CAD design using Fusion 360 and/or Meshmixer
- Apply lattice throughout the CAD geometry to create cell scaffolding using Within Medical
- Prepare for 3D printing (add supports, slicing)with Netfabb or Meshmixer
- Prepare or obtain non-toxic cell growth media for use as 3D printing resin (optional biodegradeable) I used a custom mixed Polyethylene (glycol) Diacrylate (PEGDA) hydrogel - see whitepapers linked on "resources" page.
- 3D print lattice/scaffold on microscopic level on Ember 3D printer
- Post process 3D prints / prepare for cell culture and growth
- Culture cells (I used Mesenchymal stem cells, or MSCs)
- Transfer cells to 3D prints for growth
- Create a supportive environment for cell growth with proper nutrients and allow to grow!
Step 3: Create Initial CAD Design
Create the overall general shape that you'd like in a CAD program.
Primitives like circular disks or rectangles work well.
SAMPLE FOR CELL CULTURE:
I created a disk for experimentation via
Fusion 360 > create menu > sphere (20mm wide x 5mm tall)
Save as STL
in Fusion, right click on file name (untitled) in browser tree > export > .stl
WHAT I DID FOR MY ARTWORK:
I merged workflows of graft and implant design with CAD design to create a hand design that combined 3D scan data of human hand bones and a biomimetic design inspired by anthropomorphic and osseointegration research studies. I used Memento for processing the scan data. Fusion 360 was primarily used for the CAD design, Meshmixer and 3ds Max softwares were also used.
Step 4: Process Design on Cellular Level
Open your CAD design in Within or Within Medical software to create a lattice / scaffold pattern of your choice on the cellular level. You may choose or create a design that best suits your needs.
In Within Medical, I applied a trabecular lattice using established ideal pore and beam sizes that replicates trabecular bone (aka cancellous or spongy bone). This shape not only provides a structure for the cells to grow along but also provides a pattern that could trigger stem cells to become uniquely bone cells.
The robost lattice topologies supplied with Within Medical software have been developed with cell growth in mind and are well suited for this function. This program and trabecular scaffold are commonly used for titanium osteoimplants as the cells tend to adhere to this structure for better integration within the body.
Import the initial design into Within Medical
In the left hand column, change the settings to:
Lattice target pore size = 0.45 mm
(Beam) thickness = 0.3 mm
Topology = trabecular
Surface trim = advanced
Click "Create Component Actions" in the upper toolbar
Once lattice has been generated select "File" > "Export" and choose "STL"
see the attached screenshot for details highlighted in red
*Included in this step is the .stl file of the trabecular lattice disc shown here that you may download and use.
Step 5: Prepare for 3d Printing
Once the design is established and processed down to the cellular level, the geometry needs to be prepared for 3D printing on the Ember. This requires adding supports to your model, slicing, and adding printer setting information then packaging it all up and sending it to the 3D printer.
Heal & Add Supports
Heal, Add supports and then export the model + supports as .stl
Meshmixer, Print Studio or Netfabb can be used to heal and auto generate or manually add supports to model.
This disc design is simple and does not need supports if printed flat.
My hand design was much more complicated and needed to be broken up into parts to fit within Ember's build envelope before processing (I used Alias for water tight tolerances). Each individual part was printed separately and reassembled after printing.
Slice Model and Add Printer Settings
Netfabb can be used to slice the model (necessary for very large, complex files like my hand design), Emberprinter.com or Print Studio can be used to slice the model, add information for the printer and package it in a way the printer can understand.
Add printer settings in accordance to your material and desired resolution.
To prepare trabecular model files for 3d printing in Pegda:
- Visit www.emberprinter.com and create or log into your account.
- Click “My Computer” to upload your supported trabecular model.
- Wait for the 3D viewer to load a preview of your model or click the "Next"
- Choose or input settings for the material you are printing in. I used a PEGDA hydrogel.
- Under Print Settings, click "Browse Material Browser". Find PEGDA 44 which is currently on the third page of the library.
- Click the plus button next to PEGDA 44, then you should be able to find it within your material dropdown list. Also see the screenshots in this step for settings.
- If you find these settings do not work well for you, experiment and alter the exposure settings as needed under "show advanced settings"
- Click "Next" to slice the model. The slicing may take a while depending on the size and complexity of your geometry.
- Click "Next", send the file to the Ember printer, download or save to usb drive to manually insert into the Ember you are using.
To learn more about how to use the Ember 3D printer, visit the Ember support website.
*Attached to this step is the downloadable sliced file prepared for printing on Ember in Pegda. You may save the zip file to a usb and put into your Ember. This file also includes the print settings in a text file so that you (and the Ember) can read it.
Step 6: 3D Print!
3D Print Lattice Design
I 3D printed my lattices on the microscopic level on the Ember 3D printer in PEGDA, an elastic hydrogel that supports cell attachment and is preferred for tissue engineering and regenerative medicine. Bioprinting trabecular scaffolds on the Ember in PEGDA this way enables cells to grow in orientations that mirror physiology and grow in forms relevant to the body.
Ember prints on the microscopic level (10, 25 or 50 microns), and you can control the settings with precision, which is great when you're experimenting with materials.
Ember's mechanical CAD, firmware and electronics are open source, so there is access to knowldege of how the printer works and can be adjusted for the specific needs and materials you are using. It is designed and built for research https://ember.autodesk.com/research
Polyethylene (glycol) Diacrylate (PEGDA) is a "blank slate" hydrogel that gels rapidly at room temperature in the presence of a photo-initiator and UV light, making it a great candidate for use with the Ember printer.
Polyethylene (glycol) Diacrylate (PEGDA) hydrogels are powerful tools for uncovering basic cellular biology because they are considered biologically inert and their mechanical properties can be varied.They are hydrophilic, elastic and can be customized to include a variety of biological molecules. PEGDA is an emerging scaffold for tissue engineering and regenerative medicine since polymerization can occur rapidly at room temperature and requires low energy input, has high water content, is elastic, and can be customized to include a variety of biological molecules (to incorporate cell attachment sites).
To 3D Print:
- prepare PEGDA resin
- follow printing instructions and print (!)
- remove support materials (if any)
- soak in a water bath for 3-5 minutes
- blot dry
- inspect under microscope for porosity and exposure
- adjust settings and reprint as needed
The resulting 3D print should be a 3D porous scaffold of PEGDA resin that mimics the structural conditions within actual bone.
Step 7: Post Process
The prints need to be prepared for cell culture so that they are non-toxic and sterile.
I am preparing my 3D prints for culturing Mesenchymal stem cells (MSCs) which possess the ability to differentiate into a variety of cell types, including bone cells.
- Soak prints in baths of distilled water until all of the monomer has been removed.
- Sterilize in autoclave.
Step 8: Culture and Expand Cells
Stem cells are cultured and allowed to grow and proliferate.
Culture Stem Cells
Once cells have expanded they are seeded onto the scaffolds and allowed to grow.
You may use cells of your choice that are appropriate for your experiment.
I recommend making 60-100 small sample 3D prints to test cell growth on.
*If this application is developed, the potential healthcare benefit of this workflow is that a patient's own stem cells could be obtained and used for culture and remodel onto a personalized bone graft design. The bone graft grows and matures, eventually creating a graft out of their own DNA which could be designed to be an exact fit and implanted with very low risk of rejection. Not only would it be made from the patients own cells for better integration, and without the possible complications of foreign implantation, but because it is alive it has the potential to continue growing and remodeling in the body!
*For my project, my objective is that stem cells remodel onto my design and grow into bone tissue. I am not intending to achieve an implantable design with this artwork. I hope to inspire researchers to continue to develop this line of work and artists and designers to conceive of the new things that can be made with living cells.
Step 9: Design Bioreactor
I wanted to take my piece out of the lab to show as artwork, which required a bioreactor (and makes this project even more complicated!).
The bioreactor used for this artwork was a generous gift from Exploratorium: The Museum of Science, Art and Human Perception, and Phil Ross / Mycoworks; with set-up and technical support from Chris Venter of Autodesk Bio/Nano Department. I used it in an atypical way by placing a large mass inside of it. One would probably not use an open fluid bioreactor like mine for this process.
If you intend to grow and study cell proliferation on 3D prints, a bioreactor may not be required. For cell and tissue culture the sterile environment of a laboratory setting may be all that is needed.
A custom bioreactor may be required for larger specimens or implant designs. For example, once the size of the graft is determined, a customized bioreactor can be designed and made to fit the graft and for the needs of cell growth on that shape. With this method, the exact shape of the design/print/graft can be preserved and nutrients can be delivered / metabolic waste can be removed in a targeted way. One approach is to design a bioreactor in the negative of the graft design, which functions as a mold to hold the growing tissue and deliver nutrients and gasses and evenly disperse them to the growing tissue. This also precisely retains the form of the intended graft design. See the resources section for whitepapers detailing this process.
Step 10: Allow Growth and See What Happens!
Feed your cells, check in on them, give them love. See what happens!
Step 11: Resources
3D Printed Biodegradeable Metal Bone Scaffolding Created
by Heidi Milkert https://3dprint.com/1604/3d-printed-biodegradable...
3D Printing of an Extremely Tough Hydrogel Junhua Wei, a Jilong Wang, a Siheng Su, a Shiren Wang, b Jingjing Qiu, a Zhenhuan Zhang, a Gordon Christopher, a Fuda Ning c and Weilong Cong https://www.researchgate.net/publication/28178165...
Bioceramics Articles. Advanced Ceramics
Biodegradable 3D Printed Metal Bone Scaffolding by Todd Halterman
Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow. Costa PF1, Vaquette C, Baldwin J, Chhaya M, Gomes ME, Reis RL, Theodoropoulos C, Hutmacher DW. http://www.ncbi.nlm.nih.gov/pubmed/24809431
Infiltration of mesenchymal stem cells into PEGDA hydrogel.
Yourek G, Xin X, Reilly GC, Mao JJ.
Bioreactor Cultivation of Anatomically Shaped Human Bone Grafts
Joshua P. Temple, Keith Yeager, Sarindr Bhumiratana, Gordana Vunjak-Novakovic, and Warren L. Grayson http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44765...
Custom-Made Computer-Aided-Design/Computer-Aided-Manufacturing Biphasic Calcium-Phosphate Scaffold for Augmentation of an Atrophic Mandibular Anterior Ridge
Francesco Guido Mangano, 1 , 2 , * Piero Antonio Zecca, 1 , 2 Ric van Noort, 3 Samvel Apresyan, 4 Giovanna Iezzi, 5Adriano Piattelli, 5 Aldo Macchi, 1 , 2 and Carlo Mangano 1 , 2 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44420...
Design properties of hydrogel tissue-engineering scaffolds
Junmin Zhu1,† and Roger E Marchant1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC32062...
Development and Characterization of a 3D Printed, Keratin-Based Hydrogel.
Placone JK1, Navarro J1, Laslo GW1, Lerman MJ2, Gabard AR3, Herendeen GJ3, Falco EE3, Tomblyn S3, Burnett L3, Fisher JP4. http://www.ncbi.nlm.nih.gov/pubmed/27129371
Engineering anatomically shaped human bone grafts.
Grayson WL, Fröhlich M, Yeager K, Bhumiratana S, Chan ME, Cannizzaro C, Wan LQ, Liu XS, Guo XE, Vunjak-Novakovic G. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28405...
A mathematical approach to bone tissue engineering
J.A. Sanz-Herrera, J.M. García-Aznar, M. Doblaré
A new approach to 3D printing the next generation of bone implants. Nottingham Trent University.
New super tough 3D printed hydrogel could repair damaged knees by Kira
Printability of calcium phosphate: calcium sulfate powders for the application of tissue engineered bone scaffolds using the 3D printing technique. Zhou Z1, Buchanan F2, Mitchell C3, Dunne N4. http://www.ncbi.nlm.nih.gov/pubmed/24656346
Printability of calcium phosphate powders for three-dimensional printing of tissue engineering scaffolds. Butscher A1, Bohner M, Roth C, Ernstberger A, Heuberger R, Doebelin N, von Rohr PR, Müller R. http://www.ncbi.nlm.nih.gov/pubmed/24656346
The Search for Better Bone Replacement: 3-D Printed Bone with Just the Right Mix of Ingredients
Blend of natural and man-made materials works best, study in mice shows Johns Hopkins http://www.hopkinsmedicine.org/news/media/release...
Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels
Thomas J. Hinton1, Quentin Jallerat1, Rachelle N. Palchesko1, Joon Hyung Park1, Martin S. Grodzicki1, Hao-Jan Shue1, Mohamed H. Ramadan2, Andrew R. Hudson1 and Adam W. Feinberg
Have more to add? Please post in comments below!
Step 12: What Inspired Me...
There is an intelligence beyond our understanding in the mystery of life and how the human body is formed. I have always used the body in my work, and create artwork using technology to explore with it means to be human. Over the past few years I have been working with generative art, which is when programming and mathematics defines the rules or features of artwork and creates much of the design on its own once this program is established. Nature provides the finest examples of generative art - we can look at the golden ratio in plants and humans. I was creating artwork with parametric and generative digital design to create forms, but it felt like there was something I could tap inherent with more mystery and surprises in the real world. So I looked within the body, at how cells articulate into different forms - what makes a cell become a beating heart, skin, or bone.
I felt completely drawn to bone. Bone is the structure and foundation that supports our bodies and provide protection, it stores minerals and energy, produces blood cells, plays a vital role in protecting the body against infection and enables us to move. What we don't usually think of is that bone is a very dynamic organ that is constantly remodeling and changing shape to adapt to the daily forces placed upon it. It seems so solid, but is so very much alive and constantly changing. Bones also represent the living and what is left after death. They can be both alive and exist inanimately. They are a material of life as well as a material that was historically used to make tools, accessories, art, and religious objects.
As I was exploring regenerative medicine, I was studying bones, medical implant design and additive manufacturing. I have been making 3d printed artwork of many different kinds of bones, and have now turned my focus to the human hand. The most recognizable human bones are hands and skulls. We express ourselves through our hands and use our hands as tools.
This project is emotional for me. I have been working with Riley and some very special children with upper limb differences, volunteering to provide customized low cost 3D printed Prosthetics to them through Superhero Cyborgs, Kid Mob and E-Nable. The work that I do with them inspires my designs and vice versa. I am also aligning this work as much as possible and contributing to the Luke Hand Project. Also, Lisa, my close friend from childhood has been fighting PH, a terminal illness for 8 years. She is a young, vibrant, beautiful woman with a bright spirit who could not receive a needed lung transplant because the medicine which keeps her alive caused scarring in her bone marrow, compromising blood cell production and causing excessive bleeding. They all inspire my work.
The far-reaching implications for the speculative design work that I am doing with growing bone could potentially help people like Lisa and persons with limb differences in in the future. The design of the hand was inspired by limb regeneration, tissue engineering research studies on creating biomimetic anthropomorphic hand which found crocheted ligaments and tendons mimic the features of the human hand. This research, along with light weighting prosthetics for Riley's arm with his chosen designs, inspired me to design a hand that incorporated scan data from female hand bones with a crocheted lace glove design. This speculative design is not only aesthetically interesting, but allows for a potential implant to be integrated with and connected to existing biological features.
As artwork, however, these cells are separated from the body without intention of going back into the body.
A major portion of this artwork that I'm creating is the cells used. I consider what does it mean for this specific piece to have human cells growing and proliferating outside of the body. My mother was a research scientist and I grew up in the lab with her. I feel inspired by her whenever I do this kind of work. She has passed away now, but I consider what would it mean if I could use her cancer cells in this piece? What if I used my own cells? Or a child's from their umbilical cord blood. Like in the famous case of HeLa cells, what does it mean for cells to live on outside of the body after that person has passed away?
In addition to medical and cosmetic implications, I consider the future of making things with cells as it applies to technology, art and design.
We are at an exciting time where we no longer need to turn to inanimate materials like metal, fabric, or paint to make an object. We can use actual living cells and tissue as materials to build with. The cell, "nature's building block" can be the basic structural unit.
How would the world be different if technology was grown from living organisms and life processes? What if art, design and objects we use everyday were made from living materials? What are the implications of using actual living materials to build with? What are the implications of manipulating living organisms and what are possible expressions of life that we could explore? Aside from medical and healthcare necessity, what are the fashion and beauty implications, and how we could remake ourselves as humans?
We bring our lives and our bodies to this work and being proficient in this field requires the collaboration of scientists, artists, engineers, technologists and philosophers. Even though I am focused on creating this artwork out of living bone with the fantastic team here at Autodesk right now, I am excited to see where this works leads in the future and what it means for our future. I have glimpsed some of the implications for medicine, biology, science, technology and art, and even fashion and archaeology. I am committed to continue to combine art, science and technology to explore the future of making things, and create artwork that helps us to better understand ourselves and each other. I would like to continue to make this kind of artwork and can see future potentials with adding hormones at specific times to encourage interesting things to occur, or using my own stem cells - for example.
It is exciting to make things we never thought possible to make before and I’m grateful to be at the forefront of this as Artist in Residence working with Bio/Nano team at Autodesk. Autodesk is a place where we have the technology, software and expertise to bring the fields of regenerative medicine, art and design, 3d printing on the microscopic level, reality capture, design and medical software, and materials research together to make this project possible.
I don't know what will happen from this project. Will the cells live? How will they grow? Will they follow the 3D printed form or will they proliferate off of the form in unexpected ways? It may take years for this piece to grow, the cells do not grow faster than they do in the human body. Please check back and see how this project grows!