20.109(S08):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 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 in both liquid and gel culture. The specific aspects of phenotype assayed will be collagen I and collagen II content (markers for cell type), as well as the general cell characteristic of viability.

Each pair of you will test three samples. One of these samples must consist of cells grown in monolayer culture. The other two 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 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.

In addition to designing your 3D culture experiment, you will have a chance to influence how your 2D samples are cultured. Recall from lecture that chondrocytes grown in monolayer de-differentiate to a fibroblastic phenotype over time. This is affected by several factors, including the frequency with which the cells are split. You should recommend a plan for splitting your cells, which will be carried out by the teaching faculty as closely as possible. In total, you will culture your cells for 13 days. If you collaborated with another group in designing your 3D experiment, you should agree upon the same culture conditions for your monolayer-grown cells as well. In this case, we can use the 2D culture results as a (very incomplete) reference point for how similar your culture techniques are.

Once you have decided on a plan, please tell the teaching faculty how many cells you need to initiate your cultures, in case extra animal tissue needs to be ordered. Per 3D sample, you will prepare 1 mL of alginate beads (thus a cell density of 1 million cells per mL would require 1 million cells per replicate). Per 2D sample, see the guidelines below. For all samples, you will need to make two cultures (one for viability testing, one for transcript/protein analysis), so please multiply your original number by two.

Some guidelines for 2D culture

In my experience culturing primary chondrocytes, if you originally plate 600,000 cells in a T25 flask (~9-10 mL total volume for this flask), they will be ready to be split in about 4 days. Once culture is established, cells that are split at 1:10 may become confluent again in 3-6 days, depending on how many times they have been split already (fibroblast-like cells grow more quickly than chondrocyte-like cells).

Some guidelines 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.

Optional culture additives (2D and 3D)

• Ascorbate
• ITS (insulin/transferrin/selenium)
• L-proline
• Non-essential amino acids
• Serum level used (default will be 10%)

Part 2: Reading period

If you wish, you can use any remaining time today to start thinking about your essay. You might start by reading the main editorial that you are to respond to, as well as the other relevant editorials and articles linked in the assignment description.

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, both of which will de-differentiate much less than our 2D sample. You might also comment on cell viability, if you expect it to vary among your samples.)