20.109(S08):Module 3

In this experiment, you will explore the basic principles and methods of tissue eneering. The goal of tissue engineering (also called regenerative medicine) is to repair tissues damaged by acute trauma or disease. Repair is stimulated by insertion of a porous scaffold at the wound or disease site; the scaffold may carry relevant mature or progenitor cells, and in some cases also soluble growth factors. Tissue regeneration shares many characteristics with natural tissue development, including the importance of appropriate cell differentiation and phenotype maintenance. We will examine the effect of physical and chemical manipulations to the cells’ environment on primary chondrocytes grown in both monolayer culture and in alginate beads. In particular, we will examine the viability, genotype, and protein production of these cartilage tissue cells.

I gratefully acknowledge Professor Alan Grodzinsky and several members of his lab group (particularly Rachel Miller), for their technical advice and stimulating discussions during the development of this module.

Module 3 Day 1: Start-up biomaterial engineering

Design/discussion day for students to choose parameters that will de-differentiate vs. promote phenotype of primary chondrocytes.

Module 3 Day 2: Initiate cell culture

Staff preps tissue digest day before; students prep 2D and 3D cultures (half-class at a time).

Other half of time can be diffusion modeling and NCBI bovine genome exploration exercises.

Note: 1 week between day 2 and day 3.

Module 3 Day 3: Testing cell viability

Take a portion of the cells, count and use Trypan exclusion, then prep for Live/Dead fluorescence assay and compare.

Module 3 Day 4: Preparing cells for analysis

Take portion of cells and isolate RNA, then run RT-PCR for collagen types I and II.

Rest of cells pellet with possible digestion step as well, depending on how protein assay will best work.

Meanwhile use ImageJ program to examine Live/Dead cells.

Module 3 Day 5: Transcript-level analysis

Run PCR fragments on gel and measure intensities using ImageJ.

Prepare day 1 of ELISA.

Module 3 Day 6: Protein-level analysis

Day 2 of ELISA.

Pehaps also DMMB assay for proteoglycans, but may be too much.

Module 3 Day 7: Student presentations

Module 3 Day 8: Wrap-up