20.109(S14):Design experiment (Day1)

Introduction

Today we will continue the discussion that we began in lecture about cell-biomaterial interactions and cartilage tissue engineering, with the ultimate goal of designing an experiment probing chondrocyte phenotype development and/or maintenance. Several papers on chondrocyte tissue culture and cartilage tissue engineering will be available in class, and you are also welcome to search the scientific literature on your own for further ideas.

A major part of this module will involve tissue culture; however, instead of growing cells on a 2D surface, you will grow them in 3D! During Module 2 you were exposed to general background about tissue culture (particularly in the Day 1 introduction). One topic that we didn't yet discuss is much about immortalized cells and where they come from. One familiar type of immortalized cell is the cancer cell. Tumor cells continuously divide, allowing cancer to invade tissues and proliferate. In this respect, cancer cells behave the same way in culture as in vivo, and under the right conditions cells taken from a tumor can divide indefinitely in vitro. Another type of immortalized cell is the embryonic stem cell. Embryonic stem cells are derived from an early stage embryo, and these cells are completely undifferentiated and pluripotent, which means that under the right conditions, they can become any mammalian cell type. Mouse embryonic stem cells have become a valuable research tool. They are also our go-to cell type when we do practice tissue culture in 20.109, as they grow even faster and more densely than CHO cells! You will not use embryonic stem cells during Module 3, but you will potentially use mesenchymal stem cells, which are multipotent cells that can develop into chondrocytes (among other cell types).

Normal Mouse Fibroblasts; Photographs courtesy of G. Steven Martin

Transformed Mouse Fibroblasts; Photographs courtesy of G. Steven Martin

Next time you will prepare cultures of bovine-derived cells in alginate beads according to the experimental design that you develop today.

Protocols

You will be lucky enough today to have the full lab period to complete your experimental design, as you have already completed practice (and real!) cell culture during Module 2.

Part 1: Experiment design

The overall goal of this module is to test the effect of the surrounding environment on cell phenotype. In particular, you will work with primary chondrocytes and/or mesenchymal stem cells in 3D gel culture. The specific aspects of phenotype assayed will be collagen I and collagen II transcript and protein levels (these are markers for cell type), as well as proteoglycan levels and the general cell characteristic of viability. You will be able to compare some of the data in your 3D culture experiments with control data from freshly isolated chondrocytes and stem cells.

Each pair of you will test two samples. Both samples will be grown in 3D alginate bead culture, and should have one parameter varied between them. For example, you might try changing the mechanical properties of the beads (how would you do this?), or the cell density within the beads. Be as creative as you like! If your protocol requires a new reagent or equipment to be ordered, we will do our best to get it in time. Of course, two samples is not very many for determining a trend. You are more than welcome to join up with another group or two in order to expand the range of the parameter you are testing (e.g., testing four cell densities instead of two). If everyone wants to test something different, that’s okay too.

Most of you should explore conditions for maintaining or destroying chondrocyte phenotype -- recall from lecture that chondrocytes grown without proper signals, for example in simple monolayer culture, tend to de-differentiate to a fibroblastic phenotype over time. We will also have some mesenchymal stem cells for a few groups to work with in order to investigate conditions that promote chondrogenesis. Please see the teaching faculty with your proposed experiment, as we have a limited amount of each cell type.

On that note, this year we are strongly recommending that teams of four people work together, with each of the two lab groups duplicating the same two samples. In the past, protein and proteoglycan concentrations have been too low for many groups to robustly analyze. Our recommended plan is thus that both groups test mRNA levels (which will tell us something about reproducibility), but that one group tests the rest of their sample for protein content while the other tests for proteoglycans. These larger sample sizes should improve signal. However, we will not absolutely require you to take this approach, because we have also increased our isolation scale over the past year.

One change to the module this year is that you will grow your cells for 9 days, rather than either 7 days (as last year) or 12 days (as during most past years). There are pros and cons to this timing that we will discuss further in class. The main con is less time for samples in different conditions to diverge in phenotype from each other. With that drawback in mind, we'll summarize below a few experiments that yielded the most dramatic phenotype differences in the past. The main pro of growing a shorter time appears to be

• Addition of TGF-β1 to chondrocyte culture
• MSC growth in 1% vs 2.5% low viscosity alginate
• Some alternative growth factors and additives for each cell type

We'll have more info available during class on a need-to-know basis :) But please do explore ideas on your own first!

Although you need not specify your entire plan today, you should hand in a draft plan to the teaching faculty. Plans that require reagents or equipment to be purchased are due by noon on Friday for T/R section and noon on Monday for W/F section. Plans that do not require additional reagents are due by Monday, April 22nd at 3 pm. Ultimately, your group or super-group should hand in the following information:

• Type of alginate to be used, and at what %
• Cell type to be used
• Cell density per condition (in cells/mL)
• Total number of cells needed
• Unique medium formulations or supplements to be used
• Unique systems (mechanical, electrical, etc.) to be used

Per 3D sample, you will prepare 1 mL of alginate beads (thus a cell density of 10 million cells per mL would require 10 million cells per sample, 20 million total).

As discussed in the pre-lab lecture, we recommend that you write a brief summary of at least 2-3 of the papers that you read/skimmed today in your notebook.

Materials available for 3D culture

Alginate company	Alginate name	Viscosity (reference standard)	G/M Ratio	Suggested use (%)
Sigma Aldrich	"low viscosity"	250 cps at 2%	~40:60	~1.2-3
Sigma Aldrich	"medium viscosity"	2000 cps at 2%	~40:60	~0.8-2

Collagen I and II gels can also be ordered upon request. Keep in mind that using collagen directly will confound your protein assay results (unless you devise some controls), but not the transcript-level assay results.

Standard stem cell media

• Differentiation medium
  • Hi-glucose DMEM
  • FCS and/or ITS+1 (insulin/transferrin/selenium)
  • Penicillin/Streptomycin (antibiotic)
  • Amphotericin B (antimycotic)
  • Non-essential amino acids
  • Sodium pyruvate
  • Proline (400 μM)
  • HEPES (10 mM)
  • Chondrogenic factors
    • TGF-beta1 (10 ng/mL)
    • Dexamethasone (100 nM)
    • Ascorbate (40 μg/mL)

• Expansion medium (only for your reference)
  • Low-glucose DMEM
  • 10% FCS
  • Penicillin/Streptomycin
  • Amphotericin B
  • HEPES buffer, 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
  • up to 5 ng/mL bFGF (basic fibroblast growth factor)

Standard chondrocyte media

• Growth medium
  • Hi-glucose DMEM
  • 10% FCS (or 2% FCS with ITS, for more defined media)
  • Penicillin/Streptomycin (antibiotic)
  • Amphotericin B (antimycotic)
  • Non-essential amino acids
  • Sodium pyruvate
  • Proline (400 μM)
  • HEPES (10 mM)
  • Ascorbate(20 μg/mL)

• Isolation media (only for your reference)
  • More info soon
  • Digest with pronase 1 hour, then collagenase overnight

For next time

1. Module 3, Day 2 will happen in two distinct shifts. Sign up for either the 1 pm or the 3 pm Day 2 session on that day's Talk page. If your culture requires complicated preparations, you will be asked to join the second group. Be sure to tell the teaching faculty a brief description of your plans before leaving today.
2. Familiarize yourself with the cell culture portion of Day 2 of this module. The better prepared we all are, the less likely it is that the day will run long. The hoods will be set up for you when you come in.
3. Write a two or three sentence description of your design plan and expected assay results, and post it on the Day 2 Talk page by 11 pm Tuesday or Wednesday, respectively. (Assay result expectations should be stated in a relative fashion: e.g., "we think [3D sample 1] will maintain a chondrocyte-like phenotype better than [3D sample 2], because..." You might also comment on cell viability, if you expect it to vary among your samples.) This posting will count for homework credit.

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