ArchaeBone: Recreating Bone Artifacts With Tissue Engineering

This project's goal is to connect one of the most basic of human technologies, the processing of bone into tools and items, and the cutting edge of human technology, tissue engineering. A full circle ode to us! To do this we proposed growing bone cells on a 3D printed scaffold of an ancient artifact.

Using tissue engineering skills learned over the course of a semester, combined with a mixed background of general lab knowledge, manufacturing, and engineering, a team of Cooper Union students presents to you, a bony artifact how-to!

A note, this was for a class, and we never got to finish the project, but the process is all there.

This process does NOT require a complex bioreactor! The bioreactor is a petri dish.

Items that you will need to complete this project:

• A biohood
• Centrifuge
• Incubator
• Many 10mL pipettes
• Many T75 flasks
• Petri dishes
• Centrifuge tubes
• A box of nitrile gloves
• Lots of ethanol
• A pipettor
• Cell scaffold**
• Osteoblasts
• FBS (Fetel Bovine Serum)
• DMEM
• P/S (Penicillin Streptomycin)
• Trypsin EDTA
• PBS (saline)
• Integrin
• Growth Factors

*T-Rex Skull image is from Thingiverse by Curriculum: http://www.thingiverse.com/thing:308335

**Scaffolds can be made out of many materials, and this will be addressed later on

Step 1: Culturing Cells

-Before we start anything, GLOVES AND STERILIZE.

- Sterilizing is critically important in the tissue engineering process. Contamination from chemicals or bacteria can destroy weeks of work and waste all the money put into the project. Ethanol is your new best friend; people might think you've become an alcoholic when they smell your new friend on you all the time, but it's the best thing ever. Also don't be overly concerned when you spill it pretty much all of the lab; it evaporates super quickly.

- Once sterile open the incubator you've been storing your osteoblasts in.

- We want to now check for confluence. Confluence is tissue talk for cell population density.

- You want to have >85% (approximately) confluence before passaging cells. Which means that 85% of the viewing field you see through your microscope has cells. This is important to stay on top of because confluent cells will not continue reproducing. It also good to grow as many cells as possible because they're a valuable resource which you might need.

- If NOT confluent, you need to give your cells more time to grow. So sterilize the flask, bring it to the biohood/clean bench, and aspirate the media out of it while being mindful of not scrape the bottom of the flask where the cells live. Discard your pipette. With a new pipette, fill just the bottom of the flask with new media.

- If confluent, you will need to passage your cells.

• Sterilize flask and bring it to the biohood.
• Aspirate media without scrape the bottom of the flask. Discard pipette.
• Wash cells in PBS by pipetting into the flask enough PBS to cover the bottom. Gently tilt the flask back and forth.
• Aspirate PBS and discard pipette.
• Pipette enough trypsin into the flask to cover the bottom. Tilt it back and forth.
• Sterilize flask and incubate it for 2 min.
• Aspirate the cells and trypsin, and insert them into a centrifuge tube.
• Centrifuge for 5 min at 1300 rpm (review centrifuging technique, and don't forget the counterweight.)
• Aspirate trypsin while being mindful of not taking the cell pellet. Discard pipette.
• Insert a known volume of media into the tube.
• Aspirate media and pellet to suspend the cells.
• Perform a cell count to understand your cell density per volume media.
• Distribute your suspended cells into new flasks.
• Cover the bottom of the flasks with media. Mark the flask with the date and your name. Sterilize and incubate.

Attached is the cell culture handbook we used in class. It is very thorough and an awesome resource.

Step 2: Make a Scaffold!

- For this part you can get creative! Our project's goal was to connect art from the ancient world to modern technology and fabrication. So we 3D printed a celtic knot ouroboros design that we made. We've attached an STL of that file which you can use to 3D print or if you're in the know then you can also laser cut a similar scaffold using the top view.

- You can make your scaffold out of many things as long as it is not toxic. We recommend clear PLA or acrylic. Clear is best because it is easiest to see cells on with a light microscope. Our scaffolds were made of VeroWhite, a type of acrylic. However, it is NOT clear which caused us some issues later on with imaging.

- Our scaffold has pores to create more surface area for the cells to adhere to, and encourage the ingrowth of cells. To do this I made a linear array of pores in solidworks and performed an extrude cut. We found that 1200 and 1300 micron diameter pores worked best (probably because it allowed the protein coating to attach better, but that's ahead.) We realize most 3D printers will struggle with something like this. My advice to you is to use a tiny drill bit, or a laser cutter. Or don't do pores at all.*

- Keep it small and thin. Petri dishes are small and shallow. If your scaffold is too big it will either not fit, or take up enough space such that you can't fill the dish with a sufficient amount of media. Additionally, petri dishes spill easily, so don't overfill them.

- Make it fairly flat, or cells will wash off. This build doesn't utilize a bioreactor which can hold media or cells in place.

*These results are supported by two experiments we did with arrays of cells and cell adhesion. Experiments like this are useful for getting an idea of the exact materials you're working with. Once again a reason to stay on top of passaging your cells and growing a lot of them.

Step 3: Scaffold Prep

- Our scaffold needed to be power washed and then soaked in a weak acid to be cleaned of undesired B material from the 3D printer.

- Before putting cells onto it, the scaffold must be sterilized. We soaked ours in ethanol for 30 minutes, and then let it sit in the biohood under UV light for another half hour to let it dry.

- Autoclaving is an option if you have a hardy scaffold made out of tough plastic or ceramic.

- The scaffold must also be coated in an integrin which is a protein coating that encourages cell adhesion to the scaffold.

• Integrins each have their own protocols and instructions, so follow yours carefully.
• We recommend fibronectin, or collagen because matrigel is difficult to pipette and measure.

- Let the scaffold soak in its coating for about 3 hours, and then move the scaffold into a new petri dish which cells will not like to attach to because the old dish is coated too.

Step 4: Check on Your Passaged Cells!

- Don't forget to STERILIZE

- Retrieve your flasks from the incubator.

- Check if confluent.

- If NO, change the media in the flasks, and repeat this step in a day or two.

- If YES, then perform the same cell passaging procedure from step one.

• You're going to passage portion of cells onto your scaffold, and the rest of the cells into new flasks to grow more cells for future experiments, or if things go horribly wrong.

- After passaging cover the scaffold in media, and incubate. The amount of media is a little iffy because you want to cover your scaffold completely, but not put so much in that you will deprive your cells of media. This problem is part scaffold design, and experience.

ProTip: After passaging cells are very easily moved around by fluid shear forces. To ensure cells stay on the scaffold and don't wash off as easily, pipette cell solution onto scaffold directly, and very gently. Then do not move the scaffold for 1-10 minutes. This will give the cells time to adhere.

Step 5: Waiting

- You're waiting for the cells on the scaffold surface to reach confluence. If your scaffold is clear you will be able to see this easily. If your scaffold is opaque you will need to use long exposure imaging and a camera microscope to get an idea of this. The images attached here are actually live dead assay images, but give an idea of what you'd see on an opaque scaffold.

- When the media needs to be changed it start to turn an orange-like color. Don't let it do that. That's letting cells live in their own waste. Change the media regularly until confluent.

• Attached is a picture of healthy and bad media. The healthy is purple, and the unhealthy is yellowish.

- Cells might not adhere to your scaffold ever. This is a bummer, and if this happens all you can really do is clean off your scaffold with bleach (because it is now covered in bio-waste), then water, and then soak it in ethanol. Good thing you passaged spare cells right? Go back to Step 3.

Step 6: Introducing Growth Factors

- We never actually got to this part in our project, but what you're doing now is introducing growth factors to confluent scaffold.

- This is done so the osteoblasts can start producing extracellular matrix. That's the hard stuff which bones are primarily made of and people are used to seeing.

- These growth factors will become a regular part of the media you feed your cells. Unfortunately, we have no info on the dosage necessary.

Step 7: Grow Grow Grow

- Keep refreshing media until you've grown bone across the entire scaffold surface.

- How long you do this for is up to you

- This could take about 3 weeks, so make sure you stay on top of replacing media as well passaging or freezing leftover cells in flasks.

Step 8: KILL'EM ALL

- You're satisfied with the bony artifact you've made now you can kill the cells on the scaffold.

- It is better to do this with an ethanol soak because you don't want to hold and interact with something you've soaked in bleach.

- Show off your cool bone thing, make new friends who appreciate your awesomeness and talent, perform weird rituals, etc...

- Try again with different porous networks and scaffold designs. Revive a dinosaur skeleton or recreate stone age tools!