Functions of Reagents, Cell Counting, and Experimental Design in Tissue Engineering

Problem 1: write down the functions of the following reagents.

a. 0.25% Trypsin

b. DAPI

c. Hoechst 3342

d. Propidium iodide (PI staining)

e. ALP staining

Problem 2. Are the following descriptions correct, if not, correct it.

a. DAPI can be used to stain cell nucleus.

b. Hoechst 3342 can be used to stain cell nucleus.

c. Propidium iodide can be used to stain cell nucleus.

d. The cells (cells we used in class, HeLa, MDA-MB-231) can grow on petri dish without additional treatments.

e. In class, we use warmed medium for cell culture.

f. In class, we use normal cell culture medium to freeze cells.

g. hMSC cells have higher differentiation rate after 14 days compared to 7 days.

h. In angiogenesis assay, we used growth factor reduced matrigel as an ECM. Matrigel is a synthetic biomaterial.

i. During adipogenic differentiation, differentiation rate can be calculated based on the bright field images acquired from experimental results.

j. ALP staining is the only way to identify differentiated cells.

Problem 3. Cell counting and migration rate

a. Assume all the cells are live cells. During cell passaging process, after centrifuge, 3mL fresh medium were added to cell pallet. After mixing, 100 uL cell suspension and 100 uL trypan blue were mixed. Then 10 uL were added to hemocytometer. Based on the following image, what is the cell concentration? (#/mL) How many cells in total in the pallet (cell #)? 

b. Assume all the cells are live cells. In the step (a), after adding 3 mL fresh medium, 100 uL cell suspension were mixed with 400 uL typan blue, the cells in hemocytometer were distributed like below, what is the concentration? (#/mL)

c. Describe how to calculate cell concentration using hemocytometer. Assume the dilution factor is 1. Identify which square should be counted.

d. During wound healing assay, the initial wound was measure as x1 micrometer, after 10 hours, the wound was measured as x2 micrometer. What is the migration rate? 

Problem 4. (10 pts) What is your career goal? Long-term goal and short-term goal? What skills do you need to help you achieve this goal? How tissue engineering lab course will help you to achieve your goal?

Problem 5:  A research scientist wants to investigate two different materials: collagen I and matrigel. Both of these two materials can be used as extracellular matrix (ECM) for studying angiogenesis.

In order to understand how ECM affects angiogenic process, the research scientist needs to design experiments to evaluate the performance of different ECM.

Based on our class lecture and your knowledge, design experiments to study the effects of different ECM on in vitro angiogenesis. Assume all the regents (cells, medium, biomaterials, staining reagent, and buffers) needed are available. Basic cell culture procedures (including passaging cells, freeze cells, and cell counting) do not need to be listed.

Need to include the following: 

Objective:

Hypothesis:

Methodology: 

In this section, you should include all the critical steps, including experimental design, image analysis, and statistical analysis.

Expected results and potential pitfalls.

Problem 6: Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury and bone tissue regeneration. The stiffness of extracellular matrix (ECM) is an important regulatory factor for hMSC osteogenic differentiation. As a graduate student, you are interested in studying how the substrate stiffness affects hMSC differentiation. In order to study this, you need to design experiments to evaluate the effects of substrate stiffness on hMSC differentiation.