Ishan Chatterjee Grade 9 – Fox Chapel Area High School 1

Tissue Engineering Hottest job for 21 st Century 2

Replacing diseased or injured tissues with tissue constructs designed and fabricated for the specific needs of each individual patient. What is Tissue Engineering? Cells Growth factors Scaffold Culture Implant If needed, harvest cells from patient. 3

Introduction: Stem Cells Stem Cell -- An unspecialized cell characterized by the capacity to give rise to various differentiated cell types C2C12 mus musculus (mouse) myoblast cell line – human stem cell surrogate C2C12 cells - model cell differentiation through myotube structure formation Serum Starvation (10% 1%) provides an inexpensive, easy means of inducing myogenesis 4

Introduction: Scaffolds Provide temporary framework for cell proliferation, migration, and differentiation Type/compostion varies with purpose, seeded cells, and host tissue compartment Many different materials (natural and synthetic) have been investigated for use in tissue regeneration Scaffold for Tissue Engineering 5

Introduction: Scaffolds Collagen Gel Scaffold – Structurally similar to natural extracellular matrix – Can often be processed under relatively mild conditions – May be delivered in a minimally invasive manner – Cheaper than other scaffold materials Gelatin is a hydrolyzed form of collagen Properties – Strength and elasticity – Biocompatibility – Biodegradability – Non-toxic breakdown materials 6

Objective Investigate the effect of scaffold degradation products (gelatin) on murine (mouse) myoblastic stem cell line (C2C12). Specifically, to assess the degradation product effects on cell proliferation, differentiation, and toxicity. – Toxicity & Proliferation: Effect measured by counting number of surviving stem cells after exposure to different concentrations of scaffold degradation products – Differentiation: assessed by visually evaluating and estimating myotube formation (photomicrographs) 7

Hypothesis Hypothesis Null hypothesis: Scaffold degradation products will have no significant effects on proliferation, survivorship, and differentiation of C2C12 cells in vitro Alternate hypothesis: Scaffold degradation products will have significant effects on proliferation, survivorship, and differentiation of C2C12 cells in vitro Single factor Analysis of Variance (ANOVA) + Dunnett’s test for comparing multiple treatment groups against control – Null hypothesis rejected if p-value <

Experimental Design Two Experiments (Toxicity/Proliferation and Differentiation) each with four groups, two control plus two treatment: 1.Group D (Control): no scaffold degradation product solution, contains deionized water 2.Group C (Control): no scaffold degradation product solution, contains neutralized pH solution 3.Group A: 1.0 mL (20% by volume) scaffold degradation product solution 4.Group B: 0.1 mL (2% by volume) scaffold degradation product solution 9

Materials and Apparatus 20 g gelatin (Knox “Gelatine” Packets) C2C12 murine myoblastic stem cells Deionized sterile water 100 mL graduated cylinder Test tube rack Incubator (37.0°C) µL pipette 0.1 – 1 mL pipette 1 – 10 mL pipette 70% Ethanol (for sterilization) Felt-tip marker 15 mL sterile conical tubes DMEM media (10% calf serum & 1% calf serum) contains salts, amino acids, vitamins, & glucose Sterile pipette tips 0.22 micron syringe filters + 10 mL syringe 150 mL HCl acid (0.1 molarity) 15 mL NaOH base (1.0 molarity) 200 g scales Electric kettle 75 mL culture flasks 25 cm 2 culture flasks 50 mL Trypsin-EDTA 1 mL Trypan Blue 32 mL PBS saline 32 mL 100% ice-cold ethanol 16 mL Toluidine Blue 2 Hemocytometers Light microscope Inverted microscope (with imaging capabilities) Class II Biosafety hood Labcoats, Eye Protection, Disposible Gloves Razor pH testing strips 1 mL tubes Delicate task wipes Counter 10

Procedure Preparation of Scaffold Degradation Materials 1.7g of gelatin powder was dissolved in 225mL of boiling deionized water to make a gel 2.Gelatin was sectioned into 10 g cubes with razor and left to dissolve in 35 mL of (0.01 M) hydrochloric acid for 24 hours 3.pH of solution was neutralized using NaOH and a neutral pH of 7 was verified using pH testing strips 4.Neutralized solution of HCl and NaOH was also prepared for controls 5.The degraded gelatin solution was sterilized under a laminar flow hood ultraviolet light for 30 minutes 11 Gelatin Gel (10 g cubes)

Procedure (contd.) Stem Cell Line Culture 1.1 mL aliquot of C2C12 cells from a cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask 2.Media changed with 15 mL fresh media to remove cryo- freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until 30-50% confluent (4*10 6 to 5*10 6 cells) 3.The culture was passed into 3 fresh 75mm 2 flasks in preparation for experiment 12

Procedure (contd.) Treatment Application (Proliferation and Differentiation: Day 0) 1.Sixteen 25mm 2 flasks were labeled - 8 for proliferation/toxicity, 8 for differentiation - A1, A2, B1, B2, C1, C2, D1, D2 2.Materials pipetted into each flask: 24 hours Incubation Group A (treatment) Group B (treatment) Group C (control) Group D (control) Gelatin Scaffold Solution1.0 mL0.1 mL0.0 mL Neutralized Solution0.0 mL0.9 mL1.0 mL0.0 mL Water0.0 mL 1.0 mL Media3.0 mL C2C12 Cell Suspension1.0 mL Total Volume5.0 mL Gelatin Concentration0.1%0.01%0.0% 13

Procedure (contd.) Stem Cell Line Proliferation A A1A2 B B1B2 C C1C2 D D1D2 Differentiation A A1A2 B B1B2 C C1C2 D D1D2 Photographs taken of cells fixed Day 7, serum starved day 3 Experiment Replicates Groups 14

Procedure (contd.) Media Replacement (Proliferation: Day 1 and Day 3, Differentiation: Day 1 and Day 5) 1.Media aspirated by vacuum/pipet 2.4 mL of media (and appropriate concentration of degradation materials to each group as specified in the previous table) was added. Flasks returned to incubator. Cell Counting (Proliferation: Day 2 and 5) 1.Media was removed by aspiration 2.2mL trypsin was added and aspirated immediately 3.1mL trypsin was added, flasks incubated for 4 minutes 4.Cell detachment confirmed by inverted scope viewing 5.Trypsinization quenched by addition of 4 mL media 6.1 mL of cell suspension added to sterile 1.5 mL microtubes 15

Procedure (contd.) 6.50 µL of trypan blue (dye exclusion method) added to cells 7.Hemocytometer was cleaned using 70% ethanol and delicate task wipes 8.25 µL of cell suspension pipetted onto hemocytometer 9.Cell counts were quantified Serum Starvation (Differentiation: Day 3) 1.Cells were trypsinized and suspended as before 2.4 mL of 1% serum media and selected concentrations of degradation materials were added to each group. Flasks were returned to incubator for 24 hours 16

Procedure (contd.) Cell Fixing and Staining (Differentiation: Day 7) 1.Media was removed from flasks by pipetting 2.Flasks were rinsed with2 mL of PBS saline 3.2 mL of ice-cold 100% ethanol was added to each flask 4.Ethanol was removed and excess ethanol allowed to evaporate at room temperature for 5 minutes 5.1 mL of 1% Toluidine Blue stain was added to each flask 6.Stain was removed and flasks rinsed with sterile water 17

Procedure contd. Cell Photomicrography (Differentiation: Day 8) 1.A Zeiss inverted microscope optical imaging system and connected computer was prepared for photomicrography 2.5 pictures of each flask were recorded: top right, bottom right, top left, bottom left, and center Proliferation : Day 3Proliferation: Day 6 Differentiation: Day 7 Examples of Cell Photomicrographs: 18

Procedure (contd.) Proliferation/Toxicity Differentiation Days End Scaffold Degradation Preparation and Stem Cell Line cultured Treatment Application Treatment Application – All flasks Media Replacement – All Flasks Media Replacement – A1, B1, C1, D1 Media Replacement Serum Starvation Cell Fixing and StainingCell Photomicrography Cell Count Taken – All Flasks Cell Count Taken – A1, B1, C1, D1 19

Results: Toxicity p < 0.01 [Scaffold degradation product] p < 0.01 Cells/mL ANOVA P-value: 1.49*

Results: Proliferation Assay 21

Results: Differentiation Group A (1.0 mL) – Differentiation fixed Day 7 Group B (0.1mL) – Differentiation fixed Day 7 Treatment Groups Higher Concentration Lower Concentration 22

Results: Differentiation (contd.) Group C (0.0 mL) – Differentiation fixed Day 7 Group D (0.0 mL) – Differentiation fixed Day 7 Control Groups Neutralized Solution Deionized Water 23

Conclusion: Toxicity/Proliferation Null hypothesis of no effect on stem cell survivorship was rejected for all treatment conditions (all p-values < 0.01) Significant growth effect observed for chosen concentrations of scaffold degradation materials Growth effect appears to be reversed when concentration increases above certain value – # cells in A (0.1%) significantly lower than # cells in B (0.01%) [p < 0.05] 24

Conclusion: Differentiation Based on visual assessment of myotube fusion, the null hypothesis cannot be rejected for the differentiation assay -- no significant effects on differentiation of the C2C12 cells were observed in vitro Note: The average number of fusions from each individual flask was evaluated, and not the density of the cells. 25

Sources of Error and Limitations 1.Murine stem cells may not be a perfect model of human stem cell behavior 2.Gelatin may have not been a precise model of a collagen gel scaffold 3.Visual assessment of differentiation not readily quantifiable 26

Experiment Extensions 1.Use human stem cells instead of murine stem cells 2.Use a collagen gel scaffold and collagenase in place of the gelatin and hydrochloric acid 3.Wider range of concentrations of scaffold solution to better understand relationship between concentration and toxicity/growth 4.For differentiation assay, explore the use of quantitative cell differentiation markers and techniques (anti-Myo D antibodies, RT-PCR, etc.) 27

References An Outreach Education Manual in Tissue Engineering, Pittsburgh Tissue Engineering Initiative "Gelatin." Wikipedia, the free encyclopedia.. "JoslinResearch.org: Home to the Research Division of the Joslin Diabetes Center in Boston." Diabetes Research at the Joslin Diabetes Center in Boston.. "Pittsburgh Tissue Engineering Initiative | Scaffold-Guided." Pittsburgh Tissue Engineering Initiative | Advancing Regenerative Medicine.. "Review of "Scaffolding in Tissue Engineering", by Peter X. Ma and Jennifer Elisseeff (Editors)." BioMedical Engineering OnLine.. "Stem cell - definition from Biology-Online.org." Life Science Reference - Biology Online.. "Tissue engineering." Wikipedia, the free encyclopedia.. 28

Acknowledgements Pittsburgh Tissue Engineering Initiative Mr. Mark Krotec Dr. Phil Cambell Dr. Conrad Zapanta Mr. Gregory Schubert My parents 29