ADS

Basic ADS (organotypic culture) protocol from Katie and Jenn

Preparing Dermis

 * Get dermis from Shiying
 * Incubate in KGM or DMEM (?) for 3 days to make sure no bacteria or yeast growing
 * Rinse in PBS—store in PBS for up to 3 months at 4C

Cutting Dermis

 * Prepare 3x3 black square from graph paper—attach to bottom of p150 dish
 * Spread dermis on p150 and aspirate PBS—do not let get dry
 * Examine dermis—do not want too thick or too thin—med transparency with few holes
 * Remove patches (epidermis) from dermis—can just pick off
 * Identify BMZ part of the membrane (matte side) and place shiny side up (want FB on BMZ)
 * Cut off edges
 * Stretch out dermis and cut appropriate amount of squares
 * Place scalpel down on dermis
 * Use forceps to pinch between blades and pull scalpel through forceps
 * Transfer dermis to 12-well plate—glossy side up
 * Evenly spread out dermis with forceps—tightly stretched out
 * Use pinched forceps to stretch out dermis
 * Can lift plate to ensure properly spread out
 * Dry dermis on plate for up to 1hr—leave plate open in hood

Seeding Fibroblasts

 * Get confluent plate of FB—split 1:12—use 1ml total per piece of dermis
 * Add 2ml 10%FBS DMEM to each well with dermis
 * Add 0.5ml resuspended FB to each well with dermis
 * Spin for 25 min at 1000rpm
 * Take out 0.5ml media and add another 0.5ml FB
 * Rotate plate and spin again
 * Change media daily for 5-7 days
 * FB should be growing around dermis

Lifting Dermis

 * Carefully wash forceps with soap and EtOH—flame
 * Get ADS molds and place in p60
 * Add 4.5ml 10%FBS DMEM if only lifting dermis or KGM if seeding KC
 * Carefully pick up top 2 corners of dermis from 12-well plate
 * WILL NEED TO FLIP DERMIS ONTO MOLDS
 * Flip dermis and place into center of ADS mold—drag from top edge into liquid and release when forceps edge on bottom of square
 * “Walk” dermis around edges of center square with pinched forceps
 * Make sure its evenly spread along square with no wrinkles
 * Once centered, make sure no air bubbles underneath center
 * Change media daily

Seeding Keratinocytes

 * Thaw matrigel in icy water to stay liquid
 * Get 1ml syringe and 22G needle, alcohol swab
 * Wipe top of matrigel bottle with alcohol swab
 * Flip over ADS molds so BMZ/FB side up on lid of p60
 * Use needle to get 0.7ml matrigel (for 4 ADS) want 4 drops per ADS
 * Squirt out air bubbles
 * Add 4 drops of matrigel to top of ADS—make sure evenly spread
 * Wait ~10min for matrigel to solidify
 * Flip over molds and place back in 4.5ml KGM—make sure no bubbles
 * Split KC and count cells—want 300-500x10e3 cells
 * Spin down after counting and resuspend so that you add 80ul per ADS (300-500x10e3 cells in 80ul)
 * Pipette 160ul and add continuously ½ amount (to middle mark on pipette tip) to center of dermis
 * Carefully check for air bubbles beneath ADS—DO NOT MOVE TOO MUCH—want KC to settle into dermis
 * Change media daily

Harvesting ADS

 * Prepare cryomolds—label and add OTC to fill half
 * Prepare ice bucket with dry ice
 * Get ADS and flip over molds with forceps to remove matrigel with scalpel then return to p60
 * Carefully lift dermis with forceps and place on lid of p60
 * Cut off edges of ADS—do not disturb center of ADS
 * If sectioning in half—cut on diagonal
 * Carefully lift from diagonal ends and place in cryomold, long edge closest to end (labeled end of mold)
 * Add OTC to fill rest of cryomold—make sure ADS is flat and there are no air bubbles
 * Place cryomold on dry ice and wait for it to solidify
 * Transfer cryomolds to plastic bags and store at -80C