We'll discuss these more as we go but here is a general explanation of the assignments:

• Complete readings prior to doing the self-test
• Self-test due - 2/2/15
• Experimental Plan outline due – 2/5/15
  • This is a draft of the experimental plan, in outline form, likely with a graphic
    
• Experimental Plan due – 2/9/15
  • This is a more complete, updated, and filled in experimental plan with:
    • a brief introduction that gives the relevant background and motivation for the experimental set up you are proposing,
    • a clear statement of the key experimental choices you will make (e.g. cell type, cell densities, scaffold type, culture time),
    • enough information for a reader to understand what you are planning to do easily and quickly
    • any useful or relevant diagrams
    • a timeline
    • Include any development of techniques or pre-testing of techniques you will do (and a timeline)
    • key references used to make or motivate these choices
    • Include a brief description of analysis you will do (we will develop those protocols later unless you need them right away)
      
• Proceedure Draft due – protocols due 2/16/15 but we will iterate until they are done
  • This is where you start writing detailed protocols for HOW you are actually going to do these things and I give you lots of feedback
  • Include technical risks
  • You should include any safety considerations (and MSDSs)
    
• Full Proposal due – 2/23/15 (this is a soft deadline)
  
  • This is where you revise and pull together all of the previous pieces into one document
  • Include a "plan B" for anything you think could fail
• Start culturing cells - ~ 2/18/15
  
• Order supplies – by 2/12/15
  
• Start culturing tissues - ~3/9/15
  
• Stop culturing tissues – ~4/6/15
• Drafts of pieces of the final report due throughout (see schedule)
  
• Presentations and Written reports – Finals Week

Key Product/Ordering/Money Information

Finances

• Each group will have ~$500 for their experiments.
• Cells(MSC, SMC, EC), culture media for expansion, and plastic disposables will be provided.
• I will try to help you share as much as possible (or buy into partial shares of certain items).
• Please account for needing enough supplies to practice.

Scaffolds

• Synthecon makes PGA scaffolds in tubes and disks. The tubes are made to order (so you choose ID and OD, wall thickness is ~1.2 mm) and take ~2 weeks to make so plan ahead if you want to use PGA tubes. The disks (25 packs) are fast. Prices are here.
• Becton Dickenson sells both type I collagen and Matrigel and Fisher distributes it. Matrigel is typically used at the stock concentration and is $177.62 for 5 ml (cat#: 356234). Collagen is used at various concentrations (cat#: 354236).
• I have enough Puramatrix (cat# 354250) to take a bath in and it could be available for free.
• We can also get these porous, freeze-dried collagen sponges from Collagen Matrix, Inc that come as a 4-pack of disks that fit a 12-well plate (and can be punched). They have a pore size of 150-500 um. They were $40 2 years ago and we should call to confirm the price if someone is interested.
• Others?

Cells

• Mesenchymal Stem cells (bovine) are available via isolation from a calf leg. Isolation andexpansion information, as well as more specified protocols are available.
• Smooth muscle (Human Aortic) cells will be purchased from Lonza (CC-2671)
• Endothelial cells (Human Umbilical Vein) will also be purchased from Lonza (CC-2519)
• Any others?

Bioreactors

• Please be conservative in your estimations of what you can build in such a short time and have a lot of backup plans for building, testing, failure, etc.
• I would be happy to acquire a small number of peristaltic pumps from Fisher as things we should own, but not 15 of them. We will need to discuss how you will have flow if you plan to.
• If you don't plan to build something, I suggest that you use well-plates with gentle rocking to culture your constructs.

Other Testing/Measurements

• Weight -- um, weigh your constructs
• Histological -- samples are fixed, embedded in paraffin, sliced (like in a deli-slicer) into 5 um slices and placed on slides (which you can make lots of)
  • Slides can be stained generally for cell morphology and matrix content
  • Slides can also be immunostained with antibodies specific to cell markers (or other things) of interest (e.g. smooth muscle actin, CD31, etc.)
• Microscopically -- your best bet here is to take small piece of your construct and stain the cells (for say, actin and nuclei) and look at them under the confocal microscope. this will really only work for small pieces and would be good for looking at initial seeding and morphology
• Biochemical -- Here you enzymatically digest a weighed construct (or half of one) and then perform various colorometric or fluorometric assays to assess things like collagen content and DNA content.

Mechanical Testing

• A common thing to do is to cut a ring and look at how much force it takes to stretch it. You can calibrate some sort of bending device, or use the instron (see example)
• Or really any old thing that has a force transducer (keeping in mind that if you are using something more like a hydrogel you need to keep the sample submerged, see example of fancy one).
• There are some descriptions of what people have used for hydrogels here and what kinds of things they do here.
• If you do testing in a bath you can add vasoconstrictors to that to measure activity (seeexample)
• You can also do things like blowing up the vessel like a balloon (of known pressure) but that is a bit more tricky (example) .