Diffusion Chamber System User`s Manual
Contents
1. V Bohner P Langguth J Biber H P Merkle Intestinal mucosal transport and metabolism of pentapeptides Proceed Intern Symp Control Rel Bioact Mater 21 895 1994 W Rubas N Jezyk and G M Grass Mechanism of dextran Transport across rabbit intestinal tissue and human colon cell link Caco 2 J Drug Targeting 3 15 21 1995 G I Gorodeski U Hopfer B J DeSantis R L Eckert E A Rorke and W H Utian Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors Am J Physiol 26 Cell Physiol 37 C1215 C1226 1995 W Rubas J Villagran M Cromwell A McCleod J Wassenberg and R Mrsny Correlation of solute flux across Caco 2 monlayers and colonic tissue in vitro S T P Pharma Sciences 5 1 93 97 1995 W Rubas M Cromwell T Gadek D Narindray and R Mrsny Structural Elements which govern the resistance of intestinal tissues to compound transport Mat Res Soc Symp Proc Vol 331 179 185 1994 E P Eddy C Wood J Miller G Wilson and I J Hidalgo A comparison of the affinities of dipeptides and antibiotics for the di tripeptide transporter in Caco 2 cells International Journal of Pharmaceutics 115 79 86 1995 Publication 5403 007 REV D Navicyte Diffusion Chamber System Appendix A Minimal Usable Volumes Allowing Fluid Circulation Across Open Reservoir Bottoms Snapwell Inserts and Tissue Diffusion Chambers Ha2. Directive to its products and will comply with any changes as they apply Do Not Dispose Product with Municipal Waste Special Collection Disposal Required 5 Navicyte Diffusion Chamber System Table of Contents SUBJECT PAGE NO Ts USING This GUIDE a aa a E aaia Eaa E 3 2 Introduction 2 1 Product Definitions and Descriptions seeen 4 2 1 1 Snapwell Cell Culture Insert ssessseeseesssseseesssrenees 4 2 1 2 Vertical Diffusion Chamber sss esssssseseseseeerrsrnene 5 2 1 3 Horizontal Diffusion Chamber SysteM 00cc0 6 2 1 4 Micro Reference Electrodes esseeeeesrseseeerreree 7 2 2 Diffusion Chamber System Care and Ma intenang esia enaa a eae ae hte eee 7 2 2 1 Diffusion Chamber Cleaning eese 7 8 2 3 Electrode Cleaning and Storage eecsceeceeeseeeeeeeneeees 8 2 4 Heat Block and Air Manifold Cleaning cceeeeeeee 8 3 Preparation of Buffers Culture and Storage of Cells for an Experiment 3 1 Buffer s Preparation 0 0 cc eececeeeeeeeeeeeteeeeeeeeteeeteeeeneeeee 9 3 1 1 Krebs Bicarbonate Buffer o0 ccceeeceesteeeeeteensees 9 3 1 2 Bicarbonate Buffer Stock Solution cecceeeeeeee 9 3 1 3 Preparation of Krebs Buffer From Stock SOUNS 3 einan enced eed eee atone 9 3 2 Transport Study Buffers ce eceeeeeseeeeeeeeeeeeseeeseetenees 10 3 2 1 Mucosal Apical BufferPreparation cccccee 10 3 2 2 Preparation of Serosal Basolateral Buffer 10 3 3 Culturing Cells
3. culture on a permeable support Snapwell cell culture devices consist of 12 mm diameter 0 4 micron pore sized polycarbonate or polyester membranes housed in detachable rings These two part assemblies are used together with Costar Diffusion Chamber Systems for performing a variety of physiological studies Once cell mono layer formation is complete the bottom section of the Snapwell is easily detached from its upper assembly for placement in the diffusion chamber system without cell monolayer disruption Publication 5403 007 REV D s Navicyte Diffusion Chamber System Introduction 2 1 2 Vertical Diffusion Chamber The Vertical Diffusion Chamber System can be used for transport studies on both fil ter grown cell monolayers and surgically excised tissue sections The Vertical Diffusion Chamber System includes six diffusion chambers a 12 channel Gas Manifold and a Heat Block Two general types of vertical chambers are currently available one for use with cells grown on Snapwell membrane supports and the other for use with tissues Tissue diffusion chambers are available with both circular and oblong openings depending upon the tissue type under study For example for intestinal tissue studies oblong openings can increase the effective surface area of the sample A low volume tissue chamber that reduces the amount of compound required to conduct perme ability studies is also available Up to six chambers of either type can be plac
4. 37 C for about 5 minutes Overexposure of cells to trypsin can damage cell membranes and can either kill the cells or retard their recovery Care should be taken to minimize exposure to the least amount of time required to release the cells from the flask Terminate the trypsinization process by adding about 10 mL of culture medi um to the flask Resuspend the cells by gently shaking the flask or by scraping them with a rubber policeman if necessary then shaking 6 Centrifuge the cell suspension for 5 minutes 7 Decant the supernatant and resuspend the cell pellet in 10 mL of fresh medium Divide the suspension among three new flasks adding additional medium to achieve the appropriate volume 3 4 6 Freezing Cells For Storage For additional information refer to the Corning guide for Cryogenically Storing Animal Cells To minimize mutations or viral contamination cells should be frozen relatively early in their growth cycle Prior to freezing cells have on hand a 10 DMSO solution cooled on ice as well as labeled and cooled 1 2 mL freezing vials DMSO prevents crystal formation and thus aids in maintaining cell membrane structure during freez ing To freeze cells for storage 1 2 3 Trypsinize the cells and centrifuge the suspension Resuspend the cell pellet in the ice cold 10 DMSO solution Dispense 1 mL aliquots of the cell suspension into the cooled freezing vials and seal tightly Place vials in the
5. DMSO 1 Centrifuge cells at 300 x g for 5 minutes 2 Decant supernatant and gently resuspend cells in 10 mL of complete medium 3 Transfer cells to a T 75 flask and incubate 3 4 4 Culturing Caco 2 Cells in Plastic Flasks Following thawing and or trypsinization cells require a few days to recover and resume normal growth Speed of cell division and growth depends upon cell density as well as culture conditions such as pH and timely medium replacement Monolayer formation should occur in about 7 8 days under normal conditions For culture in plas tic flasks following thawing 1 Maintain the cells in about 15 mL of medium in a T 75 flask or 30 mL of medium in a T 150 flask 2 Change the culture medium every other day by removing spent medium from the flask with a sterile pipette and replacing it with fresh medium 3 Note the color of the culture medium Color changes as pH changes While a slight color change is normal drastic color changes indicate abnormal condi tions A change toward yellow indicates acidity toward red more basicity Publication 5403 007 REV D 4 Navicyte Diffusion Chamber System Preparation and Storage 3 4 5 Caco 2 Cell Subculture To expand Caco 2 cell colonies 1 Decant medium from flask and rinse 3 times with phosphate buffered saline PBS Add trypsin solution 0 25 trypsin with 0 2 EDTA to the flask distribut ing it gently over the cell monolayer Maintain the culture at
6. This section describes the general care cleaning maintenance and storage conditions for Diffusion Chambers Heat Block Air Manifold and Micro Reference Electrodes AND CLEANERS WITH THE DIFFUSION CHAMBERS DIFFUSION CHAMBERS SHOULD NOT BE WASHED IN ALCOHOL OR AUTOCLAVED f WARNING NEVER USE ORGANIC SOLVENTS 2 2 1 Diffusion Chamber Cleaning New diffusion chamber systems require an initial washing to remove all traces of machining lubricants Ultrasonic cleaning with a detergent is also recommended after each use Diffusion chambers can be disinfected with 50 bleach or sterilized with Wescodyne Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Introduction Cleaning agents such as Ter g A Zyme made by Alconox can be used This is a con centrated anionic detergent with protease enzyme for manual and ultrasonic clean ing Excellent for removal of proteinaceous soils tissue blood and body fluids from glassware metals plastic ceramic porcelain rubber and fiberglass with no interfer ing residues Ideal as a cleaning agent in Reverse Osmosis and Ultra Filtration Systems USDA authorized Dilute 1 100 pH 9 5 2 3 Electrode Cleaning and Storage Following each use of an electrode in protein containing solutions the electrode should be soaked for 10 15 seconds in an enzyme cleaning solution or a chromic sul furic acid glass cleaning solution to remove the protein from the reference barrel and the referenc
7. center of a 10 cm2 freezer box 5 Place the freezer box in a 80 C freezer for at least two hours The tempera ture of the cells will decrease approximately 1 C per minute Vials can be left in the freezer overnight Transfer the frozen cells to the vapor or liquid phase of a liquid nitrogen stor age tank Keep the vials on dry ice during the transfer process Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Setting Up the Experiment 4 Setting Up the Experiment In any experiment the minimal usable volume of tissue culture medium that volume allowing fluid circulation across the bottom of the open reservoir should be used in the diffusion chambers See Appendix A for usable volumes The following steps should be followed for all diffusion chamber set ups 1 The Heat Block should be connected to a circulating water bath 2 The Snapwell inserts or Tissue Mounting Rings Horizontal Diffusion Chamber System only should be mounted 3 The Air Manifold should be attached to the chamber Vertical Diffusion Chamber System only These steps are described in detail below A 25 antifreeze in distilled water solution should be circulated through the external heating unit and aluminum block to prevent corrosion and to retard mold growth in the tubing Prior to use Diffusion Chambers and Heat Blocks should be preheated or cooled for about 30 40 minutes Since adding cold solutions to the tissue or ce
8. mm pin P 1mm L Gold tip Chlorided Microporouse connector silver wire ceramic tip Se p a Glass barrel 2 1 4 Micro Reference Electrodes Harvard Navicyte Micro Reference Electrodes fit both Vertical and Horizontal Chamber Systems without modification These electrodes allow measurement of the electrical parameters of tissues and cell monolayers The electrodes have a shaft diam eter of 2 5 mm and a shaft length of 5 0 cm The silver silver chloride electrode uses a refillable glass barrel that can contain any suitable electrolyte solution Usually the filling solution is the buffer used for the external medium or silver chloride AgC saturated 3 M potassium chloride KCI The glass barrel surrounds a chlorided silver wire and terminates with a ceramic tip The microporous ceramic tip limits the flux of ions into and out of the electrode and in most applications serves as a suitable replacement for bridge systems The electrode connects to a wire one meter in length which terminates in a standard Imm pin plug Snapwell Diffusion Chambers 4mm Round 9mm Round 4x8mm oblong 6x9mm oblong 8x24mm oblong and 5x24mm oblong require electrode caps to position the electrodes correctly The Low Volume and Horizontal Chambers do not require these caps Micro Electrodes are not included with either the Vertical or Horizontal Diffusion Chamber Systems and must be ordered separately 2 2 Diffusion Chamber System Care and Maintenance
9. Diffusion Chamber System User s Manual Vertical Ussing amp Horizontal Diffusion Chamber Systems MA1 66 00XX HARVARD ES Navicy Te APPARATUS Publication 5403 007 REV D WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive We will continue to monitor the application of the RoHS
10. apwell Inserts or Tissue Segments are in Place and All Connections Appropriately Made 1 Secure the front panel of the Heat Block to ensure a constant temperature 2 Sample the receiver half cell at regular intervals every 15 30 minutes for typical transport studies A typical sample volume is 1 mL but the volume can be varied depending upon the sensitivity of the assay used for analyte detection in the receiver solution The volume removed should be replaced with fresh buffer to maintain constant volume in the receptor chamber and sink conditions 3 Sample the donor side chamber at the end of the experiment 4 6 Setting Up Horizontal Diffusion Chambers For use of Horizontal Diffusion Chamber Systems some special considerations apply These systems are designed primarily as semi perfused systems in which buffer or medium is perfused through the receiver area Gas or fluid perfusion path Media perfusion path Snapwell or tissue ring Horizontal Diffusion Chamber Cross Section 4 6 1 Pumps for Horizontal Diffusion Chambers To adequately perfuse the cells or tissues in the Horizontal Diffusion Chamber System syringe or peristaltic pumps are recommended Syringe pumps deliver con stant flow without pressure changes but allow only a limited stock of donor volume and do not permit access to the donor reservoir during the pumping process Peristaltic pumps allow access to donor solution during pumping Either a common medi
11. ate to be used during the experiment Fluid will begin to accumulate in the upper chamber as pressure forces it through the membrane 5 Run the system until the rising fluid reaches a steady state level 6 Mark the fluid level in the top chamber This level is the fluid level that must be applied to the upper chamber to equalize the hydrostatic pressure Alternatively the pressure may be equalized by using the closed top configu ration and applying a gas pressure through the luer fittings 4 9 Using Electrodes with Various Chamber Types Prior to using Harvard Navicyte Micro Reference Electrodes in any configuration the reference barrel must be filled with internal reference solution To fill the barrel 1 Remove the glass barrel from the electrode cap by grasping each end of the cap and pulling until it comes apart Note The top and bottom cap sections are matched sets and should not be interchanged with other electrode sections 2 Fill the glass barrel with reference solution using the polyethylene tubing filling fiber provided with the electrode A 26 gauge or 27 gauge syringe needle will fit inside the tubing 3 Insert the silver wire into the glass barrel and reassemble the electrode cap 4 Before use place the tip of the electrode into a test tube or beaker contain ing the experimental buffer electrolyte for 1 hour or until there is no drift in measurements If little or no response occurs 1 Visually inspect the refere
12. cap place a small amount of Vaseline on the threads 4 6 5 Mounting Tissue in Horizontal Chambers To mount tissues in horizontal chambers 1 Unscrew and remove the top cap to gain access to the inside of the chamber 2 Open the optional tissue insert and place the tissue across the pins Trim the tissue so that it does not extend past the outer edge of the ring 3 Place the lower portion of the insert back on the pins then insert with the pins facing the chamber bottom 4 Replace the top cap on the chamber and tighten as above taking care not to over tighten 4 7 Fittings for Horizontal Chamber Systems Luer fittings supplied with the Horizontal Diffusion Chamber Systems are used to con nect tubing for fluid and gas flow within these chambers This flexibility permits the choice of larger tubing for higher flow rates or smaller tubing to minimize the over all volume of the receiver chamber For lower and upper chamber connections 1 The lower chamber fluid inlet can be found on the chamber front Fluid exits the chamber from the back If following the initial setup bubbles occur in the lower chamber tilt the chamber toward the front to help clear the bubbles from the back If the lower chamber is used as a stagnant reservoir a Pasteur pipette can be used in the exit channel to add media and take samples 2 The upper chamber fittings are not directional To create a completely closed system place a short loop of tubing between t
13. e Caco 2 cell culture medium add the following to Dulbecco s modified Eagle s Medium DMEM high containing 25 mM HEPES 10 fetal bovine serum 1 non essential amino acids 1 L glutamate 100 units mL penicillin 100 g streptomycin Medium components can be divided into aliquots in individual vials and frozen to facilitate complete medium preparation Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Preparation and Storage 3 4 2 Thawing Cells Prior to removing cells from liquid nitrogen prepare an ice bath a 50 mL conical cen trifuge tube and have on hand cold complete medium To thaw cells 1 Remove the cells from the liquid nitrogen storage tank placing them on ice while transferring to the laboratory Warm cells immediately to 37 C in a water bath shaking occasionally Thaw cells until no ice crystals remain Transfer cells gently to the 50 mL centrifuge tube Mix cell suspension with about 10 mL of cold complete medium o E NS Mix the medium with the cell suspension by performing 1 1 dilutions until a complete volume of medium has been added For example add 1 mL of medium to 1 mL of cell suspension and mix add 2 mL of medium and mix add 4 mL and mix etc Dilution should occur over a 2 minute time period 3 4 3 Dimethyl Sulfoxide Removal The dimethyl sulfoxide DMSO used in freezing cells must be removed from the cells carefully since it is toxic to cells at higher temperatures To remove
14. e Diffusion Chamber System Preparation and Storage 3 2 Transport Study Buffers The following buffer preparations are typically used in transport studies but may not necessarily be suitable for all applications For information regarding specific applica tions refer to the references in Section 5 3 2 1 Mucosal Apical Buffer Preparation For mucosal buffer preparation add drug or test buffer substance When glucose is used in serosal buffers 3 2 2 add an equivalent mM amount of unlabeled mannitol to the mucosal buffer The mannitol equalizes the osmotic load between the mucos al and apical buffers If the drug is radiolabeled 1 Vacuum distill the drug to remove the organic solvents 2 Distill tritiated compounds prior to use to remove any free tritium 3 Dissolve the drug in the mucosal buffer solution If the drug is unlabeled 1 Add the drug at a concentration that will dissolve completely in the buffer 2 Adjust the mannitol concentration so that the drug plus the mannitol con centration totals 40 mM For both radiolabeled and unlabeled drugs 1 Sample the mucosal buffer at the beginning of the experiment to determine the initial drug concentration 2 Sample the mucosal buffer at the end of the experiment to deter mine the decrease in donor drug concentration if any during the experiment 3 2 2 Preparation of Serosal Basolateral Buffer For Serosal Buffer preparation add 40 mM unlabeled D glucose to Krebs B
15. e eae ash ae ane 18 4 6 4 Mounting Snapwell Insets ssec 18 4 6 5 Mounting Tissue in Horizontal Chambers 18 4 7 _ Fittings for Horizontal Chamber Systems ee 18 4 7 1 Screws for Horizontal Chambers sssr 19 4 8 Controlling Hydrostatic Pressure in Horizontal Diffusion Chamber Systems cccccceeeeceeesseteeeseeeees 19 4 8 1 Equalizing Hydrostatic Pressure in Horizontal Chamber SYStOMS sisererora deirik iei 19 4 9 Using Electrodes with Various Chamber Types 19 4 9 1 Using Electrodes with Snapwell Inserts 8 x24 mm and 9 mm Tissue Chambers 20 4 9 2 Use of Electrodes with Horizontal Chambers and 9 mm Low Volume Chambers wc 21 4 10 Use of Perfusion Caps for Operation of Snapwell Inserts and Tissue Chambers excluding Low Volume Chambers for Operation in the Perfusive M d lt i a Aviiatinlind Aichi iene 21 5 Reference Literature for Tissue and Cell Culture Diffusion Chamber Systems ccceccceeeeeeeeeeeteees 22 23 6 Appendix A Minimal Usable Volumes Allowing Fluid in Circulation in Vertical Chamber Systems Snapwell Inserts and Tissue Diffusion Chambers 05 24 26 Publication 5403 007 REV D s Navicyte Diffusion Chamber System Using This Guide AND CLEANERS WITH THE DIFFUSION f WARNING NEVER USE ORGANIC SOLVENTS CHAMBERS AND THE FRONT PANEL 1 Using This Guide This User s Guide is divided into five secti
16. e junction This soaking prolongs the useful life of the electrode Always clean an electrode prior to storage Do not allow salt solutions to crystallize in the ceramic junction For long term storage longer than four weeks 1 Remove the glass barrel containing the electrolyte then store the barrel in a stoppered test tube filled with reference electrolyte or distilled water 2 Rinse the silver wire and electrode cap to remove salt solution then dry using an absorbent towel 3 Store the electrode in a closed container to prevent dust accumulation For short term storage place the tip of the electrode in a test tube or beaker contain ing reference electrolyte 2 4 Heat Block and Air Manifold Cleaning The Heat Block and Air Manifold can be rinsed with ethanol or washed with detergent Do not wash the Heat Block s acrylic front panel with ethanol or other organic solvents Publication 5403 007 REV D s Navicyte Diffusion Chamber System Preparation and Storage 3 Preparation of Buffers Culture and Storage of Cells for an Experiment 3 1 Buffer s Preparation The following section details the preparation of Krebs Bicarbonate Buffer bicarbon ate buffer stock solution and buffers for transport studies Mucosal apical and Serosal basolateral Buffers 3 1 1 Krebs Bicarbonate Buffer Krebs Buffer is a typical buffer to which test substances can be added and in which this experiment will be described Krebs Buffer
17. ed in the Heat Block at one time allowing convenient execution of six simultaneous transport studies under precise experimental control Both the vertical and horizontal described in 2 1 3 chambers permit the use of elec trodes not included with these systems but which can be purchased separately for measurement of parameters including voltage potentials and transmembrane electri cal resistance Electrode part Closed Top ia Open Top Tissue Mounting Snapwell Ring Se Heat Block Externally supplied thermostatically Electrode port Closed top Open top Tissue mounting ring Snapwell controlled H O Heat Block Externally supplied thermostatically controled H O Publication 5403 007 REV D 4 Navicyte Diffusion Chamber System Introduction 2 1 3 Horizontal Diffusion Chamber System The Horizontal Diffusion Chamber System is designed for transport and toxicology studies using cells and tissues which are exposed to an air interface in their normal in vivo environment such as nasal pulmonary corneal or dermal cells The chambers cre ate an environment in which the apical surface of the tissue or cell monolayer is exposed to liquids semi solid compounds or gases while the basolateral surface is perfused with medium The Horizontal Diffusion Chamber consists of six horizontal chambers into which either Snapwell devices or Tissue Mounting Rings may be fit ted on a horizontal plane and a Heat Block Either a peri
18. ell insert in the diffusion chamber This helps seal the system during the initialization process allowing the system to be set with a bare membrane in place 2 Place the electrodes in the chamber 3 Set offset and fluid resistances To zero electrodes for 9 mm Round and 8x24 mm Tissue Chambers 1 Open the Tissue Diffusion Chamber 2 Place a rubber band around the perimeter of the pins to seal the chamber during electrode initialization to prevent chamber leaking Alternatively Parafilm can be placed over the pins and hole the same size as the chamber opening can be cut 3 Seal the chamber 4 Place the electrodes in the chamber 5 Set offset and fluid resistances Publication 5403 007 REV D Navicyte Diffusion Chamber System Setting Up the Experiment 4 9 2 Use of Electrodes with Horizontal Chambers and Low Volume LV Chambers Horizontal Diffusion Chambers and Low Volume Tissue Diffusion Chambers do not require a cap system to position electrodes Electrodes are held in position by a screw and O ring assembly For mounting electrodes in Horizontal Diffusion Chambers 1 Place the electrode through the screw and then the O ring 2 Insert the completed assembly into the electrode port and begin to tighten the screw 3 Slide the electrode back and forth in the assembly once the O ring begins to compress As the screw is tightened the O ring begins to compress around the electrode 4 Tighten the sc
19. erfusing it through the chamber 3 Do not use the Air Manifold for perfused chamber half cells which are per fused Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Reference Literature 5 Reference Literature for Tissue and Cell Culture Diffusion Chamber Systems 10 11 12 G M Grass and S A Sweetana In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell Pharm Res 5 372 76 1988 G M Grass and S A Sweetana A correlation of permeabilities for passively transported compounds in monkey and rabbit jejunum Pharm Res 6 857 862 1989 G M Grass S A Sweetana and C Bozarth The effects of enprostil and RS 86505 007 on in vitro intestinal permeability of rabbit and monkey J Pharm Pharmacol 42 40 44 1990 N Jezyk R Kos W Rubas and G M Grass In vitro intestinal segment perme ability differences in rabbit dog and monkey Pharm Res 7 5117 1990 W Rubas N Jezyk R Kos and G M Grass In vitro transport characteristics of Peyer s patches for passively and actively transported compounds Proceed Intern Symp Control Rel Bioact Mater 1990 K L Audus R L Bartel I J Hidalgo and R T Borchardt The use of cultured epithelial and endothelial cells for drug transport and metabolism studies Pharm Res 7 435 451 1990 IJ Hidalgo K M Hilgren G M Grass and R T Borchardt Characterization of the unstirred wate
20. fold for this diffusion chamber has been specially designed for use with mixed oxygen gases Oxygen in the presence of hydrocarbons is combustible Do not use this manifold to deliver gases which contain traces of hydrocarbons Medical grade gases are recommended Other than to replace air lines do not open the Air Manifold by removing fittings as this may introduce contaminants into the oxygen cleaned environment To attach the Air Manifold 1 Attach air lines to the chambers using the holes on the outside edge at the top of each half cell Place a bubble trap between the air supply tank and the air manifold then bubble the gas through water in this trap Low humidity gas directly from the gas supply tank will cause significant fluid loss from the chamber if it is not humidified prior to delivery to the air manifold The inlet pressure to the Air Manifold should be 20 30 psi Adjust the flow to provide adequate mixing and oxygenation generally to 15 20 cc mm in each half cell but this may vary according to application or experiment 12 Channel Gas Manifold Press to Release Output Hose Flow Adjustment Locking Ring Input Pressure 20 30ps Output Pressure Generally 15 20cc min in each half cell NOTE Output pressure may vary according to application and experiment Publication 5403 007 REV D J Navicyte Diffusion Chamber System Setting Up the Experiment 4 5 Continuing the Experiment Once Sn
21. he two fittings Additionally a Pasteur pipette fits through the upper channels for sampling when the closed configuration is used Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Setting Up the Experiment 4 7 1 Screws for Horizontal Chambers Four electrode and four alternate screws without center holes are supplied with the Horizontal Diffusion Chamber System While the use of the electrode screws will be described in a later section the alternate screws for applications in which electrodes are not required prevent fluid leakage from the chamber 4 8 Controlling Hydrostatic Pressure in Horizontal Diffusion Chamber Systems Equalization of hydrostatic pressure across a membrane can be challenging in Horizontal Diffusion Chamber Systems This is of particular concern for cell monolayers because a net pressure from the basolateral to the apical side can force the cells from the mem brane To minimize the potential for hydrostatic pressure problems keep the perfusion tubing on both the inlet and outlet tubing as close to the benchtop as possible 4 8 1 Equalizing Hydrostatic Pressure in Horizontal Diffusion Chamber Systems 1 Place a bare Snapwell insert into the chamber 2 Leave the top reservoir empty of fluid 3 Connect the tubing and pump to the lower chamber and fix the tubing at the same height that will be used during the experiment 4 Pump fluid through the lower chamber at the same rate as the r
22. ing Snapwell Inserts To Mount Snapwell Inserts 1 Rinse medium from the filter with 1 2 mL of Krebs Buffer pH 7 4 2 Remove the lower filter ring from the Snapwell insert upper assembly 3 Insert the filter as shown in figure at right with the O ring in place on the chamber half cell 4 Join the matching half cell and seal the chamber with the retaining rings 5 Place the assembled chambers in the Heat Block and fill with the buffers 6 Add buffers to each side of the chamber simultaneously if possible Fill the apical mucosal side first if only one buffer can be added at a time See Appendix for appropriate half cell buffer volumes Simultaneous buffer addi tion prevents hydrostatic pressure from forcing cells off the filter surface O Ring Snapwell Publication 5403 007 REV D 7 Navicyte Diffusion Chamber System Setting Up the Experiment 4 3 Mounting Tissue Segments To mount tissue segments in the diffusion chambers 1 Mount the tissue segments on the pins of a Tissue Diffusion Chamber 2 Trim the tissue if it protrudes beyond the outer chamber sides 3 4 Again as above add buffers to each side of the chamber simultaneously fill Place the assembled chambers in the Heat Block and fill with the buffers ing the apical mucosal side first if only one can be added at a time Adding buffers simultaneously prevents hydrostatic pressure effects 4 4 Attaching the Air Manifold The Air Mani
23. lf Cell Volume mL Chamber Exposed TYPE SURFACE AREA CM Snapwell Chamber 1 13 8 x 24 Tissue 1 78 9 mm Round Tissue 0 64 9 mm LV Tissue 0 64 5 mm LV Tissue 0 20 12mm LV Tissue 1 78 Calculation of Permeability 4 0 6 0 5 0 7 0 5 0 7 0 1 0 2 0 1 5ml 0 2 0 4 2 0 4 0 Permeability coefficients may be calculated from the following equation t P V C AeC where V Volume of the receiver half cell A Exposed surface area filter or isolated tissue Initial donor concentraion C t Change in receiver concentration over time Generally a plot is made of the concentrations determined in the receiver solution versus time This is a flux curve 9c t The permeability coefficient normalizes this curve for the starting concentraion and surface area so that comparisons can be made between dissimilar experiments Publication 5403 007 REV D 2 Navicyte Diffusion Chamber System Appendix A Calculation for Sampling witb Replacement When samples are removed from the receiver chamber and replaced with blank buffer a correction must be made to account for the mass of drug the sampling procedure This correction is additive for successive samples and can be calculated as follows M mass of drug transported during the sample interval S Sample Volume removed V Total volume of the half cell receiver volume C Concentration in the chamber at the time of sampling C Corrected Co
24. ll monolayer may cause damage the temperature of buffer solutions should also be appropriately adjusted For Snapwell insert and diffusion chamber surfaces and volumes as well as perme ability calculations See Appendix A To Bath Thermocirculator 4 Barb Fitting Gas Flow Adjustment Valve Gas CO Air Manifold From Bath Thermocirculator Pump 1 4 Barb Fitting Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Setting Up the Experiment 4 1 Connecting the Diffusion Chamber Heat Block Connect the Heat Block to a circulating water bath to permit temperature control The acrylic stand provided with the units should be used in conjunction with the Heat Block to prevent radiated heat loss from the back and bottom of the Heat Block Use of a high throughput rate large heating capacity pump ensures the best temperature control and allows multiple heat blocks to run from one unit Since the Heat Block operates with high efficiency however almost any circulating water bath can ade quately maintain one complete chamber system For Vertical Diffusion Chamber Systems 1 Connect the outflow from the thermocirculator pump to the lower fitting on the back of the Heat Block 2 Connect the upper fitting on the back of the block with an 8 10 inch section of tubing to the lower fitting on the front panel of the Heat Block 3 Connect the upper fitting on the front panel back to the pump 4 2 Mount
25. ll type Typical seeding densities for Caco 2 cells are 63 000 cells cm2 for example if plating 0 5 mL into each Snapwell insert at 63 000 cells 0 5 mL adjust the concentration to 126 000 cells mL 4 Centrifuge and suspend cell pellet in the volume of medium required to obtain the desired cell concentration 5 Seed cells onto filters and add additional medium to cluster plate 0 5 mL inside 2 mL outside of each well 6 Maintain the cells at culture conditions and change medium as required usual ly every other day 3 4 Caco 2 Cell Culture General Instructions The following describes procedures for proper cell culture and maintenance General instructions for proper culture and maintenance include the following 1 Perform all procedures in a sterile environment 2 Keep the laminar flow hood clean by wiping with 70 ethanol after use 3 Keep the UV light on in the hood when hood is not in use 4 Have all supplies at hand in the hood to minimize movement in and out of the hood when the cells are out of the incubator 5 Properly dispose of all pipettes flasks vials etc that have been used to handle the cells 6 Treat spent medium with bleach and flush down the sink if appropriate 7 Following medium preparation thawing and resuspension of cells see below for specific instructions cells should be incubated at 37 C with 90 humidi ty in 5 CO2 and 95 O2 3 4 1 Cell Culture Medium Preparation To Prepare th
26. nce electrode for broken or dissolving internal Ag AgCl wire or for inadequate volume of reference electrolyte The Reference electrolyte should cover at least the Ag AgCl element 2 Soak the tip of the electrode in warm not hot distilled water for 5 10 minutes if the liquid junction is blocked or clogged An ultrasonic bath may also be used 3 Soak overnight in distilled water if still clogged or replace reference bar rel with the extra barrel supplied Publication 5403 007 REV D 5 Navicyte Diffusion Chamber System Setting Up the Experiment 4 9 1 Using Electrodes with Snapwell Inserts and Tissue Chambers These chamber types 4mm Round 4x8mm oblong 6x9mm oblong and 5x24mm oblong require a specialized electrode mounting cap There are two cap designs one for the Snapwell device and the other for tissue chambers The cap is fastened to the cham ber by a screw that fits the threaded port at the top rear of the chambers Electrodes can then be slid into the ports on the cap Electrode set up zeroing procedures depend upon the electronic device used with the electrodes While Harvard Navicyte electrodes are compatible with most commercially available voltage clamps single and six channel voltage clamps from Precision Instrument Design and Physiologic Instruments are rec ommended with the initialization procedures described in their respective manuals o To zero electrodes for Snapwell inserts 1 Mount a Snapw
27. ncentration for no dilution X C C correction factor or C C X For the initial sample Mass M V V C M V C M V Since there has been no previous dilution X 1 and C C For the second sample M M M 2V S V v So that C Moy S O y and 2M C V Therefore 2V 1 and 1 2V S 2 Publication 5403 007 REV D Navicyte Diffusion Chamber System Appendix A Subsequent samples can be calculated in a similar manner and results in the following series for 1 X Sample 1 1 X Sample 2 1 1 S gt gt X 2 V Sample 3 2 s 1 sy SEV 3 7v Sample 4 1_ 3 3 2S Beale Oy 3 7v 4 yv Sample 5 1 4 s 6s 4 ss ifs o 2 7v 3 7v 4 v 5 v The following patterns exist and make it possible to predict the correction factor for any sample number 1 Each correction factor contains a S V term raised to each power from 0 to n 1 where n is the sample number 2 The denominator for each coefficient term is equal to the power of S V 1 3 The numerator of each coefficient is equal to the corresponding number of Pascal s triangle Publication 5403 007 REV D
28. on Snapwell Filters eee 10 3 3 1 Coating Filters for Optimal Cell Adhesion ANG CURUIO erena nia a a deeds Bae 10 3 3 2 A Typical Filter Coating Procedure seses 10 3 3 3 Seeding Cells On to Filters sessssoeeseennseeeeenenenee 11 3 4 Caco 2 Cell Culture cceccccsescceeesceeeeeeeeseneeeesssessneees 11 3 4 1 Cell Culture Medium Preparation ssssssseesesseseee 11 34 2 Thawing Cells unini retai onan 12 3 4 3 Dimethyl Sulfoxide Removal ssssessssseserrnsereen 12 3 4 4 Culturing Caco 2 Cells in Plastic Flasks 12 3 4 5 Caco 2 Cell Subcultures seessseeeeeensseeeererrreneee 13 3 4 6 Freezing Cells for Storage esesesreerrrerrerrens 13 Publication 5403 007 REV D Navicyte Diffusion Chamber System Table of Contents SUBJECT PAGE NO 4 Setting Up the Experiment 4 1 Connecting the Diffusion Chamber Heat Block 15 4 2 Mounting Snapwell Inserts 0 0 eceeeeeeeeeeeeeeeeeeeteeeeeees 15 4 3 Mounting Tissue Segment cecceeeeeeseeeeeeeeteeeeeeeneees 16 4 4 Attaching the Air Manifold seeen 16 4 5 Continuing the Experiment 0eceeeeeeeeeeeeeeeeeeeeeeeeees 17 4 6 Setting Up Horizontal Diffusion Chambers 0 17 4 6 1 Pumps for Horizontal Diffusion Chambers 17 4 6 2 Using the Heat Block with Horizontal DIFFUSION Chambers oc eeeeeececcteteneteeeseeneeeeenseenees 18 4 6 3 Cap Type Selection for Horizontal CHAMDCIS ena a asia ca
29. ons and one appendix Section 1 This section describes the general organization of this User s Guide Section 2 This section is the introduction which provides descriptions and illustrations of the Costar Snapwell Cell Culture Insert the Horizontal and Vertical Diffusion Chamber Systems and the separately provided electrodes for the chamber systems This section also includes general information about diffusion chamber and electrode mainte nance Section 3 This section describes preparations for using the diffusion chambers for a model epithelial transport experiment with Caco 2 cells Specific information includes instructions for buffer preparation collagen coating of Snapwell filters cell seeding Caco 2 cell culture culture expansion cell freezing and storage Section 4 This section describes the experimental set up for using diffusion chambers with Snapwell inserts or with tissues Specific information includes the experimental set up use of the Heat Block with the chambers mounting Snapwell inserts or tissues and electrode use Section 5 This section lists selected references for specific experimental applications using tis sue and cell culture diffusion chamber systems Appendix A Includes information on minimal diffusion chamber volumes and permeability calculations Publication 5403 007 REV D Navicyte Diffusion Chamber System Introduction 2 INTRODUCTION Studies of fundamental cellular p
30. r layer in Caco 2 cell monolayers using a novel diffusion apparatus Pharm Res 8 222 227 1991 G M Grass W Rubas and N Jezyk Evaluation of CACO 2 monolayers as a pre dictor of drug permeability in colonic tissues FASEB Journal 6 4 A1002 1992 S C Sutton A E Forbes R Cargill J H Hochman and E L LeCluyse Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical resistance TEER using Sweetana Grass diffusion cells Pharm Res 9 316 319 1992 W Rubas N Jezyk and G M Grass Linear relationship between the perme ability of a human colon adenocarcinoma cell line Caco 2 and rabbit colon Proceed Intern Symp Control Rel Bioact Mater 12 306 7 1992 M E Dowty K E Knuth B K Irons and J R Robinson Transport of thyrotropin releasing hormone in rabbit buccal mucosa in vitro Pharm Res 9 1113 1121 1992 W Rubas M Cromwell T Gadek T Nguyen and R Mrsny Physicochemical properties of drugs determine diffusion across Caco 2 monolayers Pharm Res 9 S 231 1992 13 J Karlsson and P Artursson A new diffusion chamber for the determination of 14 drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer Pharm Res 9 S 180 1992 I J Hidalgo K M Hillgren G M Grass and R T Borchardt A new side by side diffusion cell for studying transport across epithelial cell monolayer
31. rew to hold the electrode in the appropriate position Do not over tighten the screw 5 Loosen the screw and slide the electrode out of the chamber without remov ing the O ring or the screw at the conclusion of the experiment 6 For use of Horizontal Diffusion Chambers without electrodes replace the electrode assembly with the screw set without the center hole with or with out the O ring For Mounting Electrodes in Low Volume Tissue Chambers 1 Place voltage passing electrodes through the two screw mounting assemblies provided with the Low Volume Tissue Diffusion Chamber 2 Place current passing electrodes vertically into the tube where air bubbles rise at the outside of the chamber to rest on the bottom of the tube 3 Loosen but do not remove the screw at the conclusion of the experiment and slide the electrode out of the chamber 4 For use of Low Volume Tissue Diffusion Chambers without electrodes a change of screw is not required 4 10 Use of Perfusion Caps for Operation of Snapwell Inserts and Tissue Chambers excluding LV Chambers for Operation in the Perfusive Mode Snapwell inserts and tissue chambers other than Low Volume chambers may be fit ted with perfusion adapters These allow continuous buffer perfusion of either or both sides of the chamber For use of Perfusion Caps 1 Fit a pumping system to the diffusion chamber similar to that described in Section 4 6 1 2 Preheat and oxygenate medium before p
32. roperties such as uptake and transport of materials across membranes and membrane electrical resistance and conductivity have been greatly facilitated by the availability of cell culture systems permitting access to baso lateral and apical cell surfaces Costar Snapwell cell culture devices and diffusion chamber systems permit convenient permeability measurements using cultured cells or tissues as models for drug absorption and transport studies Use of diffusion cham ber electrodes allows assessment of cell monolayer or tissue resistance and electro physiological properties including short circuit current measurements The following User s Guide details the use of the Costar Snapwell cell culture device in conjunction with diffusion chamber systems to perform transport and electrical studies with specific application to Caco 2 cells as a model system 2 1 Product Definitions and Descriptions This section describes Snapwell cell culture inserts the Vertical and Horizontal Diffusion Chamber Systems and Micro Reference Electrodes 4 Diffusion Chamber J Perfusion Adapter Open Top E t o ane s 1 Pi frese Diffusion is a p A Chamber me ae A g Snapwell Diffusion AA b as Optional Chamber Tissue e Mounting Ring Vertical Diffusion Chamber System 2 1 1 Snapwell Cell Culture Insert Snapwell devices are two piece Transwells which allow convenient polarized cell
33. s In Vitro Cell Dev Biol 28A 578 580 1992 Publication 5403 007 REV D 4 Navicyte Diffusion Chamber System Reference Literature 15 16 17 18 19 20 21 22 23 24 25 26 N Jezyk W Rubas and G M Grass Permeability characteristics of various intestinal regions of rabbit dog and monkey Pharm Res 9 1580 1586 1992 MJ Rutten Use of commercially available cell culture inserts for primary cul ture and electrophysiologic studies of Guinea pig gastric mucous epithelial cells J Tiss Cult Meth 14 235 246 1992 W Rubas N Jezyk and G M Grass Comparison of the permeability character istics of a human colonic epithelial Caco 2 cell line to colon of rabbit mon key and dog intestine and human absorption Pharm Res 10 113 118 1993 K Ng H Lane G Grass and R T Borchardt Characterization of the unstirred water layer in cultured brain microvessel endothelial cells In Vitro Cell Dev Biol 29A 627 629 1993 B Haeberlin H Nolen W Rubas and D R Friend In vitro studies in the evalu ation of glucuronide prodrugs for novel therapy of ulcerative colitis Proceed Intern Symp Control Rel Bioact Mater 20 172 173 1993 G M Grass C A Bozarth and J J Vallner Evaluation of the performance of con trolled release dosage forms of ticlopidine using in vitro intestinal permeabili ty and computer simulations J Drug Targeting 2 23 33 1994
34. should be sterile filtered for long term storage Shown below are recipes for stock solutions and instructions for propor tioning them to prepare the final Krebs Buffer 3 1 2 Bicarbonate Buffer Stock Solution To prepare each stock solution 1 Weigh salts 2 Add salts to a volumetric flask 3 Dissolve and dilute to the appropriate volume with reagent grade water 4 Store in refrigerator until use Stock solutions may be stored refrigerated for one week Sample Krebs Buffer Stock Solutions are given in Table 1 3 1 3 Preparation of Krebs Buffer From Stock Solutions 1 Add 50 mL of each solution in the numbered order as depicted in Table 1 above to a small volume of reagent grade water about 200 mL Mix in each solution prior to adding the next to prevent calcium salt precipitation 2 Dilute the mixture with reagent grade water to a final volume of 1 liter Final salt concentrations of salts in Krebs Buffer are shown below in Table 2 Table 1 Table 2 Krebs Buffer Stock Solutions Krebs Bicarbonate Buffer pH 7 4 Grams per Salt Final Concentration mM Solution Salt 500 mL 200 mL MgCl 1 10 Solution 1 MgCL 6H 0 2 235 0 894 CaCl 2145 CaCl 6H 0 1 835 0 734 i NaCl 114 00 Solution 2 NaCl 66 60 26 640 KCI 3 750 1 50 KCI 5 00 Solution 3 Na HPO 4 420 1 768 Na HCO 25 00 NaH PO 0 415 0 166 Na HPO 1 65 Solution 4 NaHCO 21 00 8 40 NaH PO 0 30 Publication 5403 007 REV D 5 Navicyt
35. staltic or a syringe pump not included with the system can be used to perfuse the lower surface of the Snapwell insert usually the basolateral surface The apical upper surface of the diffusion chamber can be used in either open or closed configuration the cell surfaces being more accessible for drug transport or cytotoxicity testing of liquids and semi solid materials in the open configuration In the closed configuration cells can be exposed to solutions perfused with gas or gas pressure can be applied The closed system also accepts diffusion chamber elec trodes for resistance measurements and electrophysiological studies Both open and closed tops are included with the Horizontal Diffusion Chamber System electrodes can be purchased separately Chambers can be sterilized with ethylene oxide allowing sterile cell culture under air interface conditions to be examined Publication 5403 007 REV D Navicyte Diffusion Chamber System Introduction It should be noted that due to the horizontal orientation of the cells or tissue within the horizontal device a hydrostatic pressure gradient will occur and be maintained across the cells or tissues There are methods which can minimize these pressure differences but for experiments in which such pressure may be undesirable the Vertical Diffusion Chamber System is recommended Please refer to Section 4 8 of this Users Guide for hydrostatic pressure gradient minimization techniques 2
36. uffer pH 7 4 D Glucose helps maintain tissue or monolayer viability 3 3 Culturing Cells on Snapwell Filters 3 3 1 Coating Filters for Optimal Cell Adhesion and Culture While a variety of cell types can be successfully grown and maintained on Snapwell filters without a collagen coating many cell types adhere to the filter surface more efficiently with collagen Some cell types may require substrates other than collagen for adherence and growth For specific application information please refer to the ref erences in Section 5 For cell types requiring collagen coatings a filter coating proce dure is described below in section 3 3 2 3 3 2 A Typical Filter Coating Procedure 1 Prepare a collagen solution by mixing one part rat tail collagen with three parts 60 ethanol EtOH 2 Add 100 ul collagen solution to each Snapwell filter insert making sure to cover the entire filter surface 3 Completely evaporate ethanol by drying the filters for 4 hours in a laminar flow hood with cluster plate lids slightly opened Publication 5403 007 REV D J Navicyte Diffusion Chamber System Preparation and Storage 3 3 3 Seeding Cells Onto Filters 1 Prewet coated or uncoated filters with phosphate buffer 15 minutes outside and 15 minutes inside 2 Trypsinize cells from stock centrifuge and resuspend pellet 3 Count cells and calculate concentration to seed at appropriate growth density Seeding density may depend upon ce
37. um or buffer reservoir may be used as a source for all channels or individual reservoirs may be used Medium or buffer should be oxygenated prior to pumping Publication 5403 007 REV D 3 Navicyte Diffusion Chamber System Setting Up the Experiment 4 6 2 Using the Heat Block with Horizontal Diffusion Chambers Horizontal Diffusion Chambers are heated with a circulating water bath similarly to the Vertical Diffusion Chamber Systems See Sections 4 and 4 1 of this Users Guide 4 6 3 Cap Type Selection for Horizontal Chambers Horizontal Diffusion Chamber Systems permit the use of either a closed or open con figuration on the donor upper reservoir Consider the following in choosing either configuration 1 To prevent evaporation or to maintain a constant gas content or pressure the closed cap should be used 2 To use electrodes the closed cap must be used 3 For semi solid materials or applications in which evaporation is not a con cern the open cap provides easier access to the upper chamber 4 6 4 Mounting Snapwell Inserts To mount Snapwell inserts on horizontal chambers 1 Unscrew and remove the top cap to gain access to the inside of the chamber 2 Place the Snapwell insert with the membrane area toward the bottom of the chamber 3 Replace the top cap on the chamber and tighten Do not over tighten the top cap Excessive pressure is not required to create a seal If you encounter diffi culty turning the
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