Stem Cell Culturing
Stem Cell Culturing
Introduction:
Stem cells are considered one of the primary components of successful regenerative medicine/tissue engineering. These cells have demonstrated the reproductive potential and plasticity required to regenerate viable tissue cell populations. At this point, researchers are racing to identify viable stem cell sources and to develop means to extract and employ them in therapy. In addition, stem cell lines are being developed to serve as a model for understanding their basic behaviors and therapeutic potential. It is important to remember that stem cell lines are not meant to be employed as a therapeutic cell population, regardless of their utility in the laboratory. A thorough description of cell culturing, stem cells, and analytical tools can be found in the Outreach Manual, pages 198-209.
In this activity, students are presented with cell cultures of a myoblastic mouse stem cell line (known as C2C12). They can then characterize the cells by microsocopy, and infer the conditions required to grow cells outside of a body. In addition, students are challenged to deduce the effects of an environmental variable (serum concentration) on the plasticity of these cells.
Materials and Procedure:
The growth and maintenance of this cell line is described in the Outreach Manual, pages 226-231. The basic procedure for passing the cells to initiate the experiment is as follows:
1.Remove the media from a flask of cells by pipetting (into waste beaker).
2.Add 1 mL of trypsin, gently rock the flask.
3.Immediately remove trypsin by pipetting into waste beaker.
4.Add 1 mL trypsin, rock and place in incubator for 5 minutes (this is time-sensitive, so be careful).
5.Slap the slide of flask to dislodge cells, confirm by microscopy, and immediately add 5mL of fresh media. Rock the flask to pool cells and store upright.
6.Add media to (A) 1 small culture dish, (B) 1 large dish, and (D) 1 25mm2 flask as follows:
(A) 2 ml(B) 4 ml(C) 4 ml
7.Resuspend (mix) cell suspension by pipetting, add cell suspension to each dish as follows:
(A) 1.0 ml (B) 2ml (C) 2ml
8.Return dishes to CO2 incubator.
9.The next day, remove the media (pipet) from 2 dishes that have confluent growth. Replace with 1% serum media.
10.Return to CO2 incubator.
12-well plate protocol:
- Pipet 1 mL of media into the top row of 4 wells and in the bottom row of 4 wells.
- Place a sterile PLA scaffold in well 1 of the top row (and well 1 of bottom row if directed by instructor).
- Transfer 1 mL of C2C12 cells into well 1 and 2 of top row. Pipet 0.5 ml into wells 3 and 4.
- Transfer 1mL of 3T3 cells into well 1 and 2 of bottom row. Pipet 0.5 mL into wells 3 and 4.
- On day 2, replace the media in wells 1, 2, and 3 (C2C12 cells) with 1% serum media.
To stain and fix cells:
1.Pour off the media into a beaker containing 10% bleach. Remind students to wear gloves.
2.Add 1 mL of phosphate-buffered saline (PBS). Gently swirl and pour off into the same waste beaker.
3.Add 1 mL of ice cold 100% ethanol. Swirl and poor off into a sink.
4.Allow the plates to air dry (approximately 5-10 minutes).
5.Add 1ml of toluidine blue stain. Swirl gently to cover surface.
6.Rinse out the stain with 3-5 mL of tap rinse water.
7.View cells (without cover) under 100x.
8.Cover the plates and seal with parafilm for later use.
Key questions:
- What differences are noticeable between the two cultures
2.What are the dark stained spheres within the cells?
3.What conditions must be replicated outside of the body (in culture) for cells to thrive
4.Consider the two-dimensional arrangement of cells on the plate. Why don’t the cells pile on top of each other (in three dimensions)? What type of cells might do this in culture
5.If this cell line was treated with the appropriate signals (growth factors), what types of tissue might result?
Stem Cell Passage Review Sheet
Pipetting
•Do not suck fluid up through cotton filter
•Do not touch tip or lower body of pipette to anything
•When not using pipette, keep in original sterilized wrapper
•Replace pipette when changing between different fluids
•Explain how to prevent getting bubbles in fluid
•Explain splash back problem from shooting liquid out to far
Flask Handling
•Always keep flask in upright direction
•Do not get fluid into flask cap
•Demonstrate how to rock/wash the flask
•Show which direction culture plates are upright
Description of Protocol:
1.Pipette media out of culture flask
Cells are now attached to bottom of flask and exposed to air
2.Wash culture flask with trypsin
Loosens cells/ECM/other junk
Need to wash trypsin across bottom of flask because that’s where cells are
3.Second trypsin treatment and incubation
Detaches cells from bottom of plate by degrading integrins/ECM
Time sensitive because trypsinizing cells for too long with degrade/kill cells
4.Slapping flask
Although trypsin degrades ECM, mechanical force still needed to knock the cells off the bottom of culture flask
5.Confirmation of detachment of cells
When fixed, cells were star-shaped and didn’t move when shaken
When loosened, cells are circular and floating when flask is shaken
6.Addition of media
Adding media once cells are removed stops trypsin reaction
Media contains basic nutrients for cells
7.Addition of pen/strep
Antibiotic that will kill many bacteria that happen to get into flask
8.Bovine serum
Mixture of stuff that cells need such as growth factors, signaling molecules
Different combinations change the way cells act in their environment