20.109(S08):Module 3: Difference between revisions
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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. | 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. | ||
[[Image:20.109-S08M3_culture.png|thumb|center]] | [[Image:20.109-S08M3_culture.png|thumb|center|400px]] | ||
[[20.109(S08):Start-up biomaterial engineering | Module 3 Day 1: Start-up biomaterial engineering]]<br> | [[20.109(S08):Start-up biomaterial engineering | Module 3 Day 1: Start-up biomaterial engineering]]<br> | ||
Revision as of 14:23, 28 January 2008
Module 3
Instructor: Agi Stachowiak
TA: Bahar Edrissi
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 typically 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 both in monolayer culture and in alginate beads. Specifically, we will assay 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.
Perhaps also DMMB assay for proteoglycans, but may be too much.
