20.109(S09):Start-up biomaterials engineering (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.

Protocols

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 the general cell characteristic of viability. You are free to propose one other assay if you wish, as you will likely have some extra time in the latter part of the module. One possibility is a DMMB colorimetric assay for proteoglycan content. 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, and investigate conditions that promote chondrogenesis. Please see the teaching faculty with your proposed experiment, as we have a limited amount of each cell type.

Per 3D sample, you will prepare 1 mL of alginate beads (thus a cell density of 5 million cells per mL would require 5 million cells per sample, 10 million total).

Materials available for 3D culture

Alginate company	Alginate name	Viscosity	G/M Ratio
Sigma Aldrich	"low viscosity"	250 cps at 2%	"high M"
FMC Biopolymer	Protanal LF 120M	70-150 cps at 1%	~40/60
FMC Biopolymer	Protanal LF 10/60	20-70 cps at 1%	~70/30

FMC Biopolymer alginates are samples generously donated by the company.

Collagen I and II gels are also available 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 medium

• Expansion Medium
  • 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)

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

Standard chondrocyte medium

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

Part 2: Reading period (optional)

If you wish, you can use any remaining time today to familiarize yourself with and begin the major assignments for Module 3.

For next time

1. 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.
2. Write a two or three sentence description of your design plan and expected assay results. (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.)