The Influence of Scaffoldings on C2C12 Cell Behaviors. 8 th Grade Emily Mullen St. Mary of the Assumption School

Transcription

1 The Influence of Scaffoldings on C2C12 Cell Behaviors 8 th Grade Emily Mullen St. Mary of the Assumption School

2 Regenerative Medicine Replacing injured tissues with tissue constructs fabricated for the needs of each patient.

3 Tissue Engineering Principles Cells ECM Defect Regeneration Hormones Blood Supply

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

5 Scaffold Types PLA Scaffolds popularly used in tissue engineering because of its biodegradability PCL Scaffolds A biodegradable scaffold material (neural engineering) UBM Scaffolds Urinary Bladder Matrix is an extracellular matrix (ECM) scaffold. It is now used in wound care management of partial and full-thickness wounds where conventional methods for wound care usually fail to give satisfactory results.

6 Stem Cells Cell that can produce lineages more specialized than themselves and can renew itself Spectrum of Stem Cell Behavior: totipotent embryonic cells pluripotent cells multipotent cells unipotent cells. Un-specialized cells that can differentiate into various body tissues and organs Have potential to cure or treat diseases such as heart disease, and diabetes

7 C2C12 Cell Line Subclone of the mus musculus (mouse) myoblast cell line. A common TE experimental model Differentiates rapidly, forming contractile myotomes and produces characteristic muscle proteins. Useful model to study the differentiation of nonmuscle cells (stem cells) to skeletal muscle cells.

8 Objective To compare stem cell behaviors on a synthetic and natural scaffold

9 HYPOTHESIS The decellularized matrix will promote the cell behaviors of attachment, proliferation, and differentiation. A secondary hypothesis is that cells will have a greater response to a growth factor cue on the decellularized matrix.

10 Materials PLA Scaffold Material C2C12 Cell Line Microscope Flasks Pipet Water Rubber Gloves Sterile Pipet Tips Well Plates Laboratory Aspirator DMEM Media (10% Serum) DMEM Media (2% Serum) Ethanol Trypsin Nikon inverted scope/computer interface Growth Factor, FGF2 Test Tube Rack 100 ml Graduated Cylinder Decellularized Extracellular Matrix Tongs

11 Procedure A 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 yielding a cell density of approximately 10 6 to 2x10 6 cells. *Between 200,000 and 300,000 cells are in 1mL. The media was replaced with 15 ml of fresh media to remove cryo-freezing fluid and incubated (37 C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/ml was reached. The culture was passed into 2 flasks in preparation for experiment and incubated for 2 days at 37 C, 5% CO 2. After trypsinization, cells from both of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/ml). 1 ml of the cell suspension and 3 ml of media were added to 9 well plates, creating a cell density of approximately 5.0 x 10^5 cells per flask. 1 ml of the cell suspension and 3 ml of media containing FGF-2 growth factor were added to 9 well plates, creating a cell density of approximately 5.0 x 10^5 cells per flask. The cells were incubated (37 C, 5% CO 2 ) for the remainder of the study.

12 Images were taken on day 4 Before the experiment the UBM and PLA scaffolds were created and sterilized. The UBM was spin coated in pepsin a technique that had never been done before. After the experiment, the well plates were imaged using a Nikon computer interface on three areas on the well plate.

13 Results

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20 UBM Scaffold with FGF2

21 UBM Scaffold without FGF2

22 Control with FGF2

23 Control without FGF2

24 With FGF2 Without FGF2 Cells have not formed nearly as many myotubes Myotube Formation

25 AMOUNT OF CELLS AMOUNT OF CELLS Amount of Cells Linear (Amount of Cells) 1,200,000 1,000, , ,000 1,000, , , ,000 0 Start After Trypsinization Amount of Cells 200,000 1,000,000 POINT IN EXPERIMENT

26 At Carnegie Mellon University during imaging.

27 Conclusion The results indicate the following The use of bladder decellularized extracellular matrix digested with pepsin to make into a hydrogel then spin coated onto coverslips had not been done before. In differentiation media the C2C12 readily differentiated. In the presence of FGF2 there was significant cell growth, but not myoblast differentiation. The growth factor affected the cells. There was not a significant difference in cell growth or differention between UBM or PLA matrix types. As predicted in the hypothesis, well plates that were not treated with the FGF2 growth factor formed greater myotubes than the samples treated with the growth factor in the media The hypothesis that stated that each scaffold was able to promote cell attachment, proliferation and a response to a outside growth factor will be supported.

28 Possible Limitations Due to the irregular 3D nature of the PLA construct, it could not be properly imaged. The incubator was shared so there is some risk of contamination. No cell counts were performed. Only imaged on one day. Scaffold areas not indentical No precise differention marker Cell images may not reflect the cells overall.

29 Extensions More consistent compensation and area of scaffold Employ other types of stem cells Other growth factors(sythergistic effects) Use of a differention marker

30 References A Andrews, Wyatt. "A "Holy Grail" Of Healing." CBS Evening News. CBS News, 7 Feb Web. 7 Nov B Badylak, Stephen, DVM. "Naturally Occurring Extracellular Matrix as a Scaffold for M... : Clinical Orthopedics and Related Research." LWW. Clinical Orthopaedics & Related Research, Oct Web. 07 Nov Biosci., Macromol. PLGA collagen Hybrid Mesh (b). Scaffold Design for Tissue Engineering (n.d.): The Massachusetts Institute of Technology. Web. C Christensen, Stephen. "Extracellular Matrix: Function, Components & Definition." Study.com. N.p., n.d. Web. 06 Nov D "3D Tissue Scaffolds." Regenerative Medicine. N.p., n.d. Web. 06 Nov

31 Davenport, R. John. "What Controls Organ Regeneration?" Editorial. Science Magazine 5 July 2005: 84. Science Magazine. American Association For the Advancement of Science, 5 July Web. 07 Nov E "Extracellular Matrix and Cell Adhesion Molecules." British Society for Cell Biology. British Society for Cell Biology, n.d. Web. 08 Nov "ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes." The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes. Hindawi Publishing Corporation, Web. 06 Nov "Extracellular Matrix." RightDiagnosis.com. Health Grades, n.d. Web. 08 Nov F Fang, Janet. "Scientists Regenerate Leg Muscles With Pig Bladder Tissue." IFLScience. IFL Science, 02 May Web. 08 Nov Fountain, Henry. "Human Muscle, Regrown on Animal Scaffolding." The New York Times. The New York Times, 16 Sept Web. 08 Nov H Hodde, Jason P. "Extracellular Matrix as a Strategy for Treating Chronic Wounds." American Journal of Clinical Dermatology, Apr Web. 07 Nov

32 I Inglish, Patricia. "How Extracellular Matrix Powder From Pigs and Self-Donated Blood Stem Cells Create Organs and Treat Muscular Dystrophy." HubPages. HubPages, 19 Sept Web. 07 Nov K Kelsey, Ilana. "Custom-Made Body Parts: Advances in Tissue Engineering." Science in the News. Harvard University, 30 Oct Web. 06 Nov L Layton, Julia. "Can Humans Regrow Fingers?" HowStuffWorks. How Stuff Works Health Department, 20 Feb Web. 06 Nov Lee, Rhodi. "Extracellular Matrix from Pigs Helps Regenerate Muscles of Injured Soldiers." Tech Times Regenerative Medicine. Tech Times, 04 May Web. 07 Nov M Morin, Monte. "Pig Bladders Help Wounded Humans Regrow Damaged Muscle, Scientists Say." Los Angeles Times. Los Angeles Times, 30 May Web. 07 Nov N Nauta, Allison. "Effective Delivery of Stem Cells Using an Extracellular Matrix Patch Results in Increased Cell Survival and Proliferation and Reduced Scarring in Skin Wound Healing." California's Stem Cell Agency. California Institute for Regenerative Medicine, Web. 07 Nov p Piore, Adam, and Scott Lewis. How Pig Guts Became the Next Bright Hope for Regenerating Human Limbs Discover Magazine. Discover Magazine, 26 Sept Web. 06 Nov. 201

33 S "Science Finding Ways to Regrow Human Fingers Fox News." Fox News. FOX News Network, 20 Feb Web. 07 Nov "Stem Cells, Regenerative Medicine, and Tissue Engineering." Fight Aging! N.p., 10 May Web. 06 Nov "Stem Cell Basics." What Are Adult Stem Cells? [Stem Cell Information]. US Department of Health and Human Services, 17 June Web. 06 Nov Singh, Vijai. "Extracellular Matrix." The New York Times. The New York Times, 16 Sept Web. 07 Nov "Tissue Engineering -- Biotechnology." Tissue Engineering -- Biotechnology Stocks. N.p., n.d. Web. 06 Nov T V Van Winterswijk, Peter J., MD. "Tissue Engineering and Wound Healing: An Overview of the Past, Present, and Future." MedScape, 19 Oct Web. 06 Nov W

34 Wysocki, Annette. "Interactions between Extracellular Matrix and Growth Factors in Wound Healing." Wound Repair and Regeneration. The International Journal of Tissue Repair and Regeneration, 13 Mar Web. 07 Nov Y Yong, Ed. "How Nails Regenerate Lost Fingertips." Nature.com. Nature Publishing Group, 12 June Web. 6 Sept

35 Interviews Campbell, P, Ph.D. Interviewed by E. Mullen. Central Catholic High School. 2:00 P.M., November 3, 2015 Hawranko, B. Interviewed by E. Mullen. Shadyside Place, 6:00 PM October 7, 2015 Krotec, M. Interviewed by E. Mullen. Central Catholic High School, Phone, and . This source was contacted numerous times and acted as a mentor in my experiment.

36 University Thesis Cooke, Michael John. "The Influence of the Extracellular Matrix on Cell Behaviour." Thesis. Durham University, Durham E-Theses. Durham University, 8 Sept Web. 07 Nov Mortimer, Robert. "STUDY OF EXTRACELLULAR MATRIX SCAFFOLD REMODELING IN THE PORCINE TEMPOROMANDIBULAR JOINT: UNIAXIAL COMPRESSION AND BIOCHEMICAL ANALYSIS." Master`s Thesis. The University of Pittsburgh, D-Scholarship@Pitt. The University of Pittsburgh, 4 Aug Web. 07 Nov Ramos, A., APPLICATIONS OF PLA - POLY (LACTIC ACID) IN TISSUE ENGINEERING AND DELIVERY SYSTEMS. Master s Thesis, University not Cited.

37 Online Journals Adams, Gary G., and Yu-Xin Cui. "Effects of Extracellular Matrix Proteins on Expansion, Proliferation and Insulin-producing-cell Differentiation of ARIP Cells." Scientific Research 2.4 (2009): Effects of Extracellular Matrix Proteins on Expansion, Proliferation and Insulinproducing-cell Differentiation of ARIP Cells. Scientific Researcher, Aug Web. 07 Nov Kang, Yunqing, Sungwoo Kim, Ali Khademhosseini, and Yunzhi Yang. "The Osteogenic Differentiation of Human Bone Marrow MSCs on HUVEC-derived ECM and β-tcp Scaffold." Biomaterials (2012): Science Direct. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 15 July Web. 8 Nov

38 Sheng, Zhiyong, and Xiaobing Fu. "Dedifferentiation: A New Approach in Stem Cell Research." Oxford Journals BioScience Talks 57.8 (2007): Oxford Journals BioScience. Oxford Journals. Web. 7 Nov