Cell Culture o The ability to grow cells mammalian amphibian bacteria plant etc in an artificial environment o 30 billion dollar industry b c biologics Cell Biology Cell Culture Molecular Biology Biologics a drug protein generated by cell cultures Most are antibodies some are receptors o Terminology In vitro in cell culture o History In vivo in the living organism In situ in place in the cell 2 start points 1900 Golgi stain defined brain tissues as cells We cannot predict cell action in vitro vs in vivo sometimes stem cell therapy issues Take brain tissue put in cell culture dish w blood clot something growing out of explant of grain tissue to support neuron theory o First time tissue grown outside of organism 1951 George Gay from Johnns Hopkins Took cells from Henrietta Lacks who had cervical cancer due to HPV worked We got lucky because we did not know how to make cells grow in culture o One of the first cell lines o Power advantages of cell culture Single cell type rather than a heterogeneous population Ability to control the environment Recombinant DNA technology biologics We can make transgenic cells can be used for biologics GFP reporter gene Stem cell therapy requires expansion propagation in cultures New stem cell technologies make stem cells Tissue engineering requires cell cultures Basic gene functions requires oncogenes Drug discovery testing o Limits concerns of cell culture Cells change their genotype over time unstable genetic makeup HeLa cells lose genes make more genes Environment of the cells is artificial You do not know if what you re looking at is representative of in vivo environment Cells don t necessarily express proper genotype e g MDCK important kidney cell line and CACO 2 intestinal ED cells o Not polarized taken out put on device w microporous membrane o Microvilli polarized apical top basal bottom components of cell o Cells do not express proper type Some cells require complex biological microenvironments e g human embryonic stem cells hESCs o feeder layer dead mouse cells required for the hESCs to grow on top of Some cells lose function over time P 1 e g human hepatocytes liver Cell Biology Cell Culture Molecular Biology o Follow daily function of hepatocytes of liver in culture someone died graph Figure out function of hepatocytes cytochrome p450 detoxifies Does function drop off if cells die Cell viability may look very well except cytochrome p450 drops off hard o Paradox hepatocytes in vivo divide but in vitro do not lose function Categories 1 Cell type Cell strain vs cell line Cell strain From isolated tissue o Take tissue EGTA protease cells put into dish primary culture o Expand tissue put into two separate dishes secondary cultures Problem doublings limited to 40 60x discovered by Leonard Hayflick o 20 years later we found out it was due to the shortening of telomeres o Inverse relationship between donor age the of potential doubles Examples o NHEK Normal Human Epidermal Keratinocytes o Hepatocyes Human Neonatal Hepatocytes PROS In vivo like CONS 40 60x limit Cell Line Immortal unlimited of doublings Embyronic cell lines o 3T3 from mouse fibroblasts Transformed cell lines 1 Cell isolated from a tumor e g PC 3 prostate cancer cell line 2 UV light chemical mutagens EMS 3 Spontaneous mutant 4 Transfect w oncogene gene that produces onco protein normal cell becomes cancer cell 5 Manipulate telomeres most common way to manipulate cell line Telomerase immortalized cell lines PROS lots of cells cancer drug CONS not normal cells 2 Growth media requirements Serum Supplemented Media Often add 10 fetal bovine serum o Contains growth factors o Adding unknowns o Animal proteins problem can carry viruses Defined Serum Free Media P 2 Cell Biology Cell Culture Molecular Biology Critical for clinical applications Serum of Youth parabiotic mice made to share blood system leg muscle injured regenerated only in young young and young old mice 3 Growth substrates Cell culture dishes o e g one well or 384 wells o 384 wells used for high thoroughput screening use to see drugs that are active on cells Beads o One bead w little cells all around them Beads put into dish w media going in through tube device at other end collects biologic protein you want to harvest o Microcarrier beads designed for high cell density o Good for generating biologics molecule protein generated by a cell type Roller bottles o Sideways bottle that you roll around use for blood cells Tubes o Sideways tube w cells that line the sides use for arterial endothelial cell May be used to observe plaque formation Feeder layers o Used for human embryonic stem cells o Mouse cells stem cells grow on them Microporous membranes o Mock cells express the proper phenotype Tissue Engineering Regenerative medicine o Uses cells cell culture to fabricate engineered tissues o Began around 1980s picture of mouse w human ear o Biological Scaffolds Biological Scaffold s Biodegradable o Over time enzymes secreted by cell biodegrade o Tissue construct w no scaffold grown Non biodegradable No need for scaffold 3D printer Stem Cells o Tissue Engineered Constructs TECs Human bladders e g adult deprived mesenchymal stem cells any cell type Spina bifida children have defective bladders autologous self transplant ELAD Extracorporeal Liver Assist Device Someone w liver problems blood goes through ELAD C3A inside toxins are gone filter device FDA C3A human hepatoma cell line is inside cartridge P 3 Cell Biology Cell Culture Molecular Biology o About 50 cyto p450 activity Caritcel Genzyme Corp Artificial cartilage knee hole transplant Autologous transplant Bronchial Vaginal TECs Toxicity testing Engineered Skin Applications Clinical burn victims diabetic ulcers Non clinical product safety testing EpiDerm skin TEC mater corp o Microporous membrane as a substrate Process 4 step process 1 Microporous membrane put on surface the right kind of microenvironment extracellular matrix primarily type 1 collagen 2 Seeding throwaway cells NHEKs put cells on surface Increase external concentration of calcium cells begin to differentiate Breast reduction surgeries Foreskin better 3 Differentiate 4 Air liquid interface How good is it Factors to consider Lowered media level exposed to media barrier o Structural analysis LM TEM good looks like skin o Biochemical signature that tells people that it is TRULY skin o Skin specific keratins using western blots good o Function o Barrier compare EpiDerm w cadaver skin bad o 50 EpiDerm leaky done in