20.109(S08):Module 3: Difference between revisions

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[[20.109(S08):Start-up biomaterial engineering | Module 3 Day 1: Start-up biomaterial engineering]]<br>
==Module 3==


Design/discussion day for students to choose parameters that will de-differentiate vs. promote phenotype of primary chondrocytes.
'''Instructor:''' [[User:AgiStachowiak| Agi Stachowiak]]


[[20.109(S08):Initiate cell culture| Module 3 Day 2: Initiate cell culture]]<br>
'''TA:''' [[Bahar Edrissi]]


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


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


Note: 1 week between day 2 and day 3.
[[Image:20.109-S08M3_culture.png|thumb|center|450px]]


[[20.109(S08):Testing cell viability| Module 3 Day 3: Testing cell viability]]<br>
[[20.109(S08):Start-up biomaterials engineering (Day1)| Module 3 Day 1: Start-up biomaterials engineering]]<br>
[[20.109(S08):Initiate cell culture (Day2)| Module 3 Day 2: Initiate cell culture]]<br>


Take a portion of the cells, count and use Trypan exclusion, then prep for Live/Dead fluorescence assay and compare.
Note: 1 week between day 2 and day 3.
 
[[20.109(S08):Preparing cells for analysis| Module 3 Day 4: Preparing cells for analysis]]<br>
 
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 final protein assay chosen.
 
Meanwhile use ImageJ program to examine Live/Dead cells.
 
[[20.109(S08):Transcript-level analysis| Module 3 Day 5: Transcript-level analysis]]<br>
 
Run PCR fragments on gel and measure intensities using ImageJ.
 
Prepare day 1 of ?ELISA? (?Western?)
 
[[20.109(S08):Protein-level analysis| Module 3 Day 6: Protein-level analysis]]<br>
 
Day 2 of collagen ELISA or Western.
 
If not possible, perhaps DMMB assay for proteoglycan instead.  
 
[[20.109(S08):Student presentations| Module 3 Day 7: Student presentations]]<br>


[[20.109(S08):End-of-term party| Module 3 Day 8: End-of-term party]]<br>
[[20.109(S08):Testing cell viability (Day3)| Module 3 Day 3: Testing cell viability]]<br>
[[20.109(S08):Preparing cells for analysis (Day4)| Module 3 Day 4: Preparing cells for analysis]]<br>
[[20.109(S08):Transcript-level analysis (Day5)| Module 3 Day 5: Transcript-level analysis]]<br>
[[20.109(S08):Protein-level analysis (Day6)| Module 3 Day 6: Protein-level analysis]]<br>
[[20.109(S08):Wrap-up analysis (Day7)| Module 3 Day 7: Wrap-up analysis]]<br>
[[20.109(S08):Student presentations (Day8)| Module 3 Day 8: Student presentations]]<br>

Latest revision as of 08:45, 29 January 2008


20.109(S08): Laboratory Fundamentals of Biological Engineering

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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 biomaterials engineering
Module 3 Day 2: Initiate cell culture

Note: 1 week between day 2 and day 3.

Module 3 Day 3: Testing cell viability
Module 3 Day 4: Preparing cells for analysis
Module 3 Day 5: Transcript-level analysis
Module 3 Day 6: Protein-level analysis
Module 3 Day 7: Wrap-up analysis
Module 3 Day 8: Student presentations

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