1 / 10

Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs

Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs. Jacqueline Mimnaugh , RET Neuqua Valley High School Dr. Richard Gemeinhart Melanie Köllmer UIC Department of Biopharmaceutical Sciences Tracy Chuong , REU University of California Berkley.

larue
Download Presentation

Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs Jacqueline Mimnaugh, RET Neuqua Valley High School Dr. Richard Gemeinhart Melanie Köllmer UIC Department of Biopharmaceutical Sciences Tracy Chuong, REU University of California Berkley

  2. hMSCs and Tissue Engineering Human Mesenchymal Stem Cells (hMSCs) Can differentiate into bone, cartilage, fat, and other cells Tissue Engineering ↓ Bone diseases/defects, trauma, cancer, mal-union/non-union fractures Osteogenesis → Induce hMSCs to develop into bone cells

  3. The Problem Cells in the lab are typically cultured on plates 2 D Cells in vivo (in an organism) 3 D It is possible that cells grown in a 3D scaffold would: • Be more like cells in vivo • Would not reach confluence as quickly • Could be implanted directly

  4. Objective: Compare the osteogenic differentiation of cells in 2D and 3D culture systems. 2D Culture Plate 3D Superporus Hydrogel Viability? Compare Cells: Proteins? Mineralization?

  5. Creating a 3d Scaffold Superporous Hydrogels • Poly (ethylene glycol) diacrylate or PEGDA • Polymer network, hydrophilic • Pores from 100 µm to 600 µm created by gel-foaming

  6. Project Overview 1. Seed hMSCs onto 2D plates and 3D hydrogels After 24 hours 2. Add osteogenic differentiation medium After 24 hours 3. Compare cells at day 2, 7, 14, 21 and 28

  7. Comparing the cells 1. MTS – Cell Viability 2. BCA – Protein Levels 3. Calcium 4. Alkaline Phosphatase – Early Marker 5. ELISA: Osteopontin – Mid-phase Marker Osteocalcin – Late Marker 6. qPCR – Determine Gene Expression - ALPL, RUNX2, OC, OP, BMP2 7. Von Kossa Staining - Mineralization

  8. MTS Results Cell Viability/Proliferation – Cells breakdown MTS into a product that can be detected by a plate reader higher absorbance = more viable cells Considerations: • Cell seeding number • Cells from SPH lost over time • Deviation – two donors

  9. ALP Results Alkaline Phosphatase – ALP is a byproduct of osteoblast activity. ALP cleaves a substrate to product a fluorescent product higher fluorescence = more ALP activity Considerations: • Normalized with MTS • Early marker of differentiation • Day 21 – Time

  10. Von Kossa Results Day 7 Day 21 Day 28 Von Kossa stain binds to phosphate and calcium in the sample More dark spots = more mineralization 2D SPH

More Related