BE 167L Lecture 8 General

BE 167L Lecture 8 General - BE 167L – Bioengineering...

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Unformatted text preview: BE 167L – Bioengineering Lab Lecture 8 For Lab •  Developing your own lab protocol for pa=erned cell culture! Today •  Co ­culture: cell cultures with mulGple cell types – Building Gssues and understanding signaling – IsolaGng soluble vs. contact factors •  SelecGve cell adhesion: pa=erning mulGple cell types •  Pa=erning DNA and proteins  ­ Microarrays Next Tuesday Case Study 6: ECM microarrays Why Co ­culture? •  Building Gssues engineered •  Uncover intercellular signaling mechanisms Soluble Factors •  CondiGoned Media –  Contains autocrine/paracrine factors at high levels –  Contains waste –  Will not allow invesGgaGon of factors that change dynamically with Gme –  Only one ­way signaling •  Transwell Plates –  Transfer of autocrine/paracrine factors – transport should be controlled –  Dynamics can be invesGgated with care –  Two ­way signaling can be invesGgated * Note: no cell-cell contact signaling with these methods Types of co ­culture •  Soluble factors only (Transwell) •  Cell ­cell contact – Random – Micropa=erned Bhatia et al. FASEB J 1999 “Random” mixed cultures Important Interactions Cell-cell interface -Homotypic vs. Heterotypic Interactions Cell ratio # / # OR * Note: Cannot be independently controlled Pa=erned mixed cultures Controlled cell ratio, different heterotypic / homotypic ratios Bhatia et al. FASEB J 1999 Differences in Albumin Expression Day 6 Day 1 Monoculture Co-culture DifferenGal adhesion to ECM for co ­culture Fukuda et al. Biomaterials 2006 mESC + hepatocyte co ­culture Made with a DNA microarraying robot Lee et al. Integrative Biology 2009 Co ­culture differenGaGon FITC - collagen Fibronectin Alkaline Phosphatase Embryonic stem cell spreading • ES cells showed hepatocyte like transcription profiles •  QuesGons raised by these experiments? DNA Microarrays Affymetrix – photolithographic DNA Microarrays Polymerase Chain ReacGon Temperature Primers Taq Polymerase Nucleotides (dATP, dTTP, dCTP, dGTP) ...
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