Stem Cell/Yeast Seeding Efficiency

Tissue Engineering Summer Camp Activities: Tissue Malfunctions/Potential Therapies

Introduction:

Stem cells are often considered the fundamental tools of regenerative medicine. They have the potential to grow into various tissues, providing biomedical researchers and clinicians with an important new type of therapy. These cells might one day be used to regenerate tissues that have been compromised by trauma, disease or old age. Some regenerative medicine researchers use stem cells to seed scaffolds outside of the body (in vitro). A scaffold is a temporary structure that is used to grow stem cells in a damaged or missing area of tissue. This cell/scaffold construct can then be transplanted into a damaged tissue site. Other researchers attempt to directly implant stem cells into the body (in vivo). It is hoped that the implanted or injected cells will survive their transfer, evade immune responses, and occupy the damaged target tissue. Ideally, these cells would then stimulate the regeneration of healthy tissue.

In this activity, gauze is used to simulate a damaged tissue (muscle) that is the target of stem cell therapy. A strain of normal-colored yellow yeast is used to represent the population of typical tissue cells. A strain of pink-colored yeast is used to represent injected stem cells. Cell seeding and survival efficiency will be addressed by the use of standard microbiological techniques. Background material and a more difficult version of this activity are presented in the Teacher Manual, Chapter 6.2, pages 239-242.

Objectives:

1. Students will master microbiological techniques and common fluid transfer techniques. 2. Students will learn to apply mathematics to the concepts of serial dilution. 3. Students will learn to quantify colonies and determine % seeding efficiency.

Materials:

Yeast cultures (wild type yellow strain, ade-2 mutant pink strain), 70% ethanol in jars, spreader bars, burners (or matches), YEPD media, YEPD-agar plates, incubator, vortex, micropipettors, sterile micropipette tips, test tubes with 9.0 and 9.5 mL of sterile distilled water, sterile gauze, long forceps or probe.

In this simulation, the yeast populations are first grown to a density of approximately 50 Klett units or an absorbance of 0.5 (at 600nm). This represents a cell density of approximately 107 cells/mL. By adding 1.0 mL of the yellow yeast to tube 1 (containing 9.0 mL sterile water), the density of cells in that tube is now 106 cells/mL. If all of the cells adhered to the tissue model (gauze), this would represent a quantity of 107 cells. 0.1 mL of pink yeast (stem cells) are then added, representing a maximum of 106 cells that could potentially adhere to the simulated tissue. Tissue Engineering Summer Camp Activities: Tissue Malfunctions/Potential Therapies

Notice also that a series of tubes is used to quantify the amount of cells that adhered to the gauze. This is done to find a concentration of cells that is easily countable on a YEPD agar plate. It is assumed that the initial tube used to dislodge cells from the gauze contains a very high concentration of cells. Therefore, a series of dilutions are performed to yield a countable number of yeast upon plating. The actual number of adhered cells can then be determined by multiplying the number of colonies by the appropriate dilution factor. For example, if 100 colonies resulted from plating 0.1 mL of the second tube after the vortexed tube (tube C), then the original vortexed tube (A) contains a concentration of, 100 x 10 x 10 x 10 = 105 cells/mL. The total number of cells adhering to gauze would then be, 105 x 10 = 106 cells.

Procedure: Stem Cell Seeding Efficiency Simulation

1. Label 5 test tubes of sterile water (1, A, B, C, D). 2. Label 4 plates (A, B,C, D) and put your name and date on each plate. 3. Add 1.0 ml of Yellow yeast to test tube 1. 4. Add 0.1 ml of Pink yeast to test tube 1. 5. Use sterilized scissors to cut a sterile gauze pad into quarters (1/4”) or less. 6. Using sterilized forceps, place the piece of gauze into test tube 1. 7. Let the test tube sit for 5 minutes. 8. Using sterilized forceps, remove the sterile gauze pad and place it in test tube A. 9. Place test tube A in a vortex to “knock off” the cells from gauze. 10. Use a micropipette (with new tip) to remove 1.0 ml of liquid from test tube A and put it in test tube B. 11. Vortex test tube B (to evenly suspend cells). 12. Use a micropipette (with a new tip) to remove 1.0 ml of liquid from test tube B and put it in test tube C. 13. Vortex test tube C (to evenly suspend cells). 14. Use a micropipette (with new tip) to remove 1.0 ml of liquid from test tube C and put it in test tube D. 15. Vortex test tube D (to evenly suspend cells). 16. Use a micropipette (with new tip) to remove 0.1 ml of liquid from test tube A and place it on plate A. 17. Use a micropipette (with new tip) to remove 0.1 ml of liquid from test tube B and place it on plate B. 18. Use a micropipette (with new tip) to remove 0.1 ml of liquid from test tube C and place it on plate C. 19. Use a micropipette (with new tip) to remove 0.1 ml of liquid from test tube D and place it on plate D. 20. Place all 4 plates in an incubator.

Calculation:

1. Count the yellow colonies on the most ‘countable’ plate. Count the number of pink colonies (NOTE: the pink color might not appear for 4-5 days). Determine the ratio of pink colonies to total colonies. Tissue Engineering Summer Camp Activities: Tissue Malfunctions/Potential Therapies

2. Determine the total number of pink colonies from your most countable tube (as evidenced by the related plate). Remember to account for the volumes of fluid involved; you added only 0.1 mL of fluid from that tube to the plate to obtain colonies. Working backwards through the dilution series, determine the total number of pink colonies that adhered to the gauze (tissue). Determine the seeding efficiency by dividing that number (pink yeast on gauze) by the total number of pink yeast loaded into the original tube.

Figure 1: Removing a simulated tissue (gauze) from a suspension of simulated stem cells (yeast)

Figure 2: Transferring the seeded target tissue to sterile fluid tubes Tissue Engineering Summer Camp Activities: Tissue Malfunctions/Potential Therapies

Figure 3: Quantifying seeding efficiency of stem cells (red yeast) amidst tissue cells (white yeast)

Figure 4: Resulting colonies from a dilution series of seeded cells.