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. You will also get some practice with cell culture today, to prepare you for beginning your experiment next week.

Background

Keeping any of this in intro? Saved some primary enough during M2 I think

Tissue culture was developed about 100 years ago as a method for learning about mammalian biology, and since that time we have learned a tremendous amount by studying the behavior of mammalian cells maintained in the laboratory. The term tissue culture was originally coined because people were doing exactly that, extracting tissue and letting it live in a dish for a short time. Today, most tissue culture experiments are done using cells rather than tissues. Much of what we know about cancer, heritable diseases, and the effects of the environment on human health has been derived from studies of cultured cells.

What types of cells do people study, and where do they come from? Cells that come from a tissue are called primary cells, because they come directly from an animal. It is very difficult to culture primary cells, largely because primary cells that are placed in culture divide only a limited number of times. This limitation on the lifespan of cultured primary cells, called the Hayflick limit, is a problem because it requires a researcher to repeatedly remove tissues from animals in order to complete a study. Cell isolation processes can be quite labor-intensive, and also can complicate data analysis due to inherent animal-to-animal variation. To get around this problem, people have studied cells that are immortal, which means that they can divide indefinitely. Some inherent cell-to-cell variation still exists in such populations. Moreover, the genetic changes that cause immortality may affect experimental outcomes.

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 culture. 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, and it is this cell type that we will be using for our practice cell culture today.

Normal Mouse Fibroblasts; Photographs courtesy of G. Steven Martin

Transformed Mouse Fibroblasts; Photographs courtesy of G. Steven Martin

The art of tissue culture lies in the ability to create conditions that are similar to what a cell would experience in an animal, namely 37°C and neutral pH. Blood nourishes the cells in an animal, and blood components are used to feed cells in culture. Serum, the cell-free (and clotting-factor free) component of blood, contains many of the factors necessary to support the growth of cells outside the animal. Consequently, serum is frequently added to tissue culture medium, although serum-free media (also called chemically defined media) exist and support some types of cultured cells.

Cultured mammalian cells must grow in a germ-free environment and researchers using tissue culture must be skilled in sterile technique. Germs double very quickly relative to mammalian cells. An average mammalian cells doubles about once per day whereas many common bacteria can double every 20 minutes under optimal conditions. Consequently, if you put 100 mammalian cells and 1 bacteria together in a dish, within 24 hours you would have ~200 unhappy mammalian cells, and about 100 million happy bacteria! Needless to say, you would not find it very useful to continue to study the behavior of your mammalian cells under these conditions!

One major objective for this experimental module is for you to learn how to perform tissue culture. Today you will learn how to get mouse embryonic stem cells to grow in a dish and also how to prevent contaminants from getting into your cell cultures. Next time you will set up 3D cultures of bovine-derived cells in alginate beads.

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: Practice cell culture

Part 2: 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 7 days instead of 12 days. There are pros and cons to this timing that we will discuss further in class, the main con being less time for samples in different conditions to diverge in phenotype from each other. With that drawback in mind, we'll summarize here a few experiments that yielded the greatest dramatic phenotype differences in the past:

• 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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