Difference between revisions of "20.109(S09):Start-up biomaterials engineering (Day1)"

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(New page: {{Template:20.109(S09)}} <font color=red>Changes from S08 to mae: use of MSCs or CDRs or both allowed; possibly no 2D sample, three 3D instead (maybe one or two groups focus on 2D if they...)
 
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<font color=red>Changes from S08 to mae: use of MSCs or CDRs or both allowed; possibly no 2D sample, three 3D instead (maybe one or two groups focus on 2D if they want to); one or two groups maybe can pilot MSC transfection studies if interest; buy some non-alginate gel materials also</font color>
 
  
 
==Introduction==
 
==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.
+
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==
 
==Protocols==
Line 11: Line 9:
 
===Part 1: Experiment design===
 
===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.
+
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 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.  
+
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, thats 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.
+
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.
  
−
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.  
+
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).  
  
−
====Some guidelines for 2D culture====
+
====Materials available for 3D 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====
+
  
 
{| border="1"
 
{| border="1"
Line 50: Line 44:
 
FMC Biopolymer alginates are samples generously donated by the company.
 
FMC Biopolymer alginates are samples generously donated by the company.
  
−
====Optional culture additives (2D and 3D)====
+
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 &mu;M)
 +
**HEPES (10 mM)
 +
**Chondrogenic factors
 +
***TGF-beta1 (10 ng/mL)
 +
***Dexamethasone (100 nM)
 +
***Ascorbate
 +
 
 +
====Standard chondrocyte medium====
  
−
*Ascorbate
+
*Growth medium
−
*ITS (insulin/transferrin/selenium)
+
**Hi-glucose DMEM
−
*L-proline
+
**10% FCS (or less, with ITS)
−
*Non-essential amino acids
+
**Penicillin/Streptomycin
−
*Serum level used (default will be 10%)
+
**Amphotericin B
 +
**Non-essential amino acids
 +
**Sodium pyruvate
 +
**Proline (400 &mu;M)
 +
**HEPES (10 mM)
 +
**Ascorbate(20 &mu;g/mL)
  
−
===Part 2: Reading period===
+
===Part 2: Reading period (optional)===
  
−
If you wish, you can use any remaining time today to start thinking about your [[20.109(S08):Essay|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.
+
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==
 
==For next time==
  
 
#Familiarize yourself with the cell culture portion of [[20.109(S09):Initiate_cell_culture_%28Day2%29 | 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.
 
#Familiarize yourself with the cell culture portion of [[20.109(S09):Initiate_cell_culture_%28Day2%29 | 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.
−
#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.)
+
#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.)

Latest revision as of 19:33, 28 July 2015


20.109(S09): Laboratory Fundamentals of Biological Engineering

20.109(S09) frontpg.JPG

Home        People        Schedule Spring 2009        | Assignments        Lab Basics        | OWW Basics       
Protein Engineering        Expression Engineering        Cell-Biomaterial Engineering              

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.)
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