Tissue/Cell Culture

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Tissue/Cell Culture

Vertical Ussing and Horizontal Diffusion Chamber System

Figure 1: Diffusion Chamber Systems

• The standard assay for oral bioavailability

• Applications include:

- Drug delivery - Toxicology - Pharmacokinetics - Metabolic studies - Transport

• US Patented technology numbers:

5, 183, 760 and 5,591,636

• Available in vertical and horizontal system configurations

• Options for using cultured cells or tissue samples

• Manufactured entirely of acrylic for reduced drug absorption/easy cleaning

• Compact configurations

• Up to 24 chambers can be run simultaneously

• Systems are temperature controlled

• Electrode ports connect optional electrodes for monolayer resistance measurements and electrophysiological determinations

The Harvard/NaviCyte patented line of horizon- tal and vertical diffusion chambers is designed for the study of substance transport across cul- tured cell monolayers or excised tissue under dynamic conditions. Figure 1 shows the basic system architecture for both the horizontal and vertical diffusion chamber systems.

Optional electrodes provide the ability to make monolayer resistance electrophysiological measurements. An optional electrical manifold can be connected to either the vertical or horizontal chamber system to allow easy connection to our various voltage/current clamp systems. The following pages describe the Vertical and Horizontal Diffusion Chamber Systems, Snapwell^™cell culture inserts and the Micro-Reference Electrodes.

Snapwell^™Cell Culture Inserts

Snapwell^™devices are two-piece Transwells^™which allow convenient polarized cell 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 the horizontal and vertical diffusion sys- tems for performing a variety of physiological studies. Once cell monolayer 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.

9 mm Vertical Diffusion Chamber

BS4 66-0023 Electrode Manifolds

Two styles of electrical manifolds are available to connect electrodes to our Voltage/Current clamp systems, see the following pages.

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Vertical Ussing/Diffusion Chamber Systems

BS4 66-0048 6 Vertical Chamber System with Gas Manifold and Heating Block

The vertical diffusion chamber system is used for transport studies on both filter grown cell monolayers and surgically excised tissue sections. The vertical diffusion chamber system consists of six diffusion chambers, a 12-channel gas manifold and a heat block. 14 general types of vertical chambers are currently available: one for use with cells grown on Snapwell^™membrane supports and the others for use with tissue.

Gas Manifold

The 12-channel gas manifold distributes gas (user supplied) to the Diffusion Chambers where media circulation is generated by gas lift, reducing unstirred fluid layers adjacent to cells.

Heat Block

The dual heating blocks of the Vertical Chamber System provide effi- cient and precise heating or cooling of the Diffusion Chamber System.

Temperature is controlled by use of a circulating water bath. The Heat Block and Gas Manifold allows six simultaneous tests to be run, improving experimental precision.

Electrode Caps

Electrode caps permit proper positioning of electrodes within the vertical perfusion chambers. They are required for the Snapwell^™ BS4 66-0008, 8 x 24 mm Tissue BS4 66-0013 and 9 mm Round BS4 66-0014 chambers. They are not required for the low volume chambers or the horizontal tissue chambers.

Perfusion Caps

The BS4 66-0019 perfusion cap allow the tissue chambers to be perfused with media using a peristaltic or syringe pump. They are required for use with the Snapwell^™BS4 66-0008, 8 x 24 mm Tissue BS4 66- 0013, and 9 mm Round BS4 66-0014 chambers. They are not required for the low volume chambers or the horizontal tissue chambers.

• Designed for easy placement of Snapwell^™(grown cells) or tissue sections

• All Snapwell^™and tissue diffusion chamber config-urations are compatible with the vertical system, providing versatility and convenience for correlation studies

• Side-by-side orientation prevents a hydrostatic pressure gradient across cell layer

• Distributes gas (user supplied) to the diffusion chambers where media circulation is generated by gas lift, reducing unstirred fluid layers adjacent to cells

• Individual control valves on gas manifold allow independent adjustment of gas flow into each diffusion chamber half-cell

• Closed perfusion capable

Adjustable Flow Valve

Heat Block Tubing to

Diffusion Chamber

Diffusion Chamber O-Ring

Snapwell

Figure 2: Vertical Diffusion Chamber System

Gas Manifold

Externally supplied O₂/CO₂

Externally supplied thermostatically- controlled H₂O

We offer 14 different vertical diffusion chambers. We also offer a complete system for each chamber that includes the following components:

Catalog No. Qty. Product

BS4 66-0009 1 6-Chamber Heating Block BS4 66-0010 1 Clear Front Panel BS4 66-0048 1 12-Channel Gas Manifold BS4 66-0012 1 Retaining Ring Clamp Tool Specified 6 Vertical Diffusion Chambers

Vertical Ussing/Diffusion Chamber Systems Catalog No. $ Chamber Included Standard Vertical Chamber Systems

BS4 66-0003 Six BS4 66-0014 - 9mm Round Chambers BS4 66-0037 4,571.00 Six BS4 66-0036 - 4mm Round Chambers BS4 66-0002 Six BS4 66-0013 - 8 x 24mm Oblong Chambers BS4 66-0033 4,571.00 Six BS4 66-0032 - 4 x 8mm Oblong Chambers BS4 66-0039 Six BS4 66-0038 - 6 x 9mm Oblong Chambers BS4 66-0047 4,571.00 Six BS4 66-0046 - 5 x 24mm Oblong Chambers Low Volume Vertical Chamber Systems

BS4 66-0028 4,571.00 Six BS4 66-0026 - Low Volume 5mm Round Chambers

BS4 66-0004 Six BS4 66-0015 - Low Volume 9mm Round Chambers

BS4 66-0029 4,571.00 Six BS4 66-0027 - Low Volume 12mm Round Chambers

BS4 66-0041 Six BS4 66-0040 - Low Volume 3mm Round Chambers

BS4 66-0035 4,571.00 Six BS4 66-0034 - Low Volume 2 x 10mm Oblong Chambers

BS4 66-0043 Six BS4 66-0042 - Low Volume 4 x 8mm Oblong Chambers

Specialty Vertical Chamber Systems

BS4 66-0001 Six BS4 66-0008 - Snapwell^™Chambers BS4 66-0045 4,571.00 Six BS4 66-0044 - 12mm Millicell^®Chambers

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Tissue/Cell Culture

Vertical Ussing/Diffusion Chambers

These chambers are designed in three formats for work with excised tissue segments. Tissue is affixed between the opposing faces of the half-chambers using a series of pins surrounding the opening.

Tissue diffusion chambers are constructed as two half-cells that are held together by a high tension spring retaining ring that insures leak free oper- ation. They are available with both circular and oblong openings, depending on the tissue type under study. For example, for intestinal tissue studies, oblong openings can increase the effective surface area of the sample.

Several low volume tissue chambers that reduce the amount of com- pound required to conduct permeability studies and custom chambers are also available. Up to six vertical chambers of any type can be placed in the heat block at one time, allowing convenient execution of six simultaneous transport studies under precise experimental conditions. The compact configuration, 38.1 x 10.2 x 12.7 cm (15 x 4 x 5 in), conserves valuable bench space. An optional heat block that can hold up to 24 chambers is also available. An optional adapter (perfusion cap) can be inserted into each half cell for closed perfusion applications.

Snapwell^™Diffusion Chambers

This chamber is specifically designed for use with the Snapwell^™ culture insert (Corning Costar Corp.). The chamber accepts the lower section of the Snapwell, which contains the tissue culture treated membrane with cell monolayer. These Snapwell^™chambers give better results than other chambers. Their low profile reduces the unstirred water layer thus more closely approaching the real conditions in the intestine.

• 14 Different chambers available in 3 configurations:

– Standard Chambers – Low Volume Chambers – Specialty Chambers

- Snapwell^™ - Millicell^®

Standard Diffusion Chamber BS4 66-0013,0032,0038,0046

Standard Diffusion Chamber BS4 66-0014, 0036

Low Volume Diffusion Chamber BS4 66-0026, 0040

Low Volume Diffusion Chamber BS4 66-0015

Low Volume Diffusion Chamber BS4 66-0027

Low Volume Diffusion Chamber BS4 66-0034, 0042

Snapwell^™Diffusion Chamber BS4 66-0008 Vertical Ussing/Diffusion Chambers

Exposed Working Tissue Reservoir

Catalog No. $ Chamber Type Area Volume

Standard Vertical Chambers

BS4 66-0014 9 mm Round 0.64 cm² 5 to 7 ml BS4 66-0036 468.00 4 mm Round 0.12 cm² 5 to 7 ml BS4 66-0013 8 x 24 mm Oblong 1.78 cm² 5 to 7 ml BS4 66-0032 468.00 4 x 8 mm Oblong 0.29 cm² 5 to 7 ml BS4 66-0038 6 x 9 mm Oblong 0.46 cm² 5 to 7 ml BS4 66-0046 468.00 5 x 24 mm Oblong 1.15 cm² 5 to 7 ml Low Volume Vertical Chambers

BS4 66-0026 468.00 5 mm Round 0.20 cm² 0.2 to 0.4 ml BS4 66-0015 9 mm Round 0.64 cm² 1 to 2 ml BS4 66-0027 468.00 12 mm Round 1.13 cm² 2 to 4 ml BS4 66-0040 3 mm Round 0.07 cm² 1 to 2 ml BS4 66-0034 468.00 2 x 10 mm Oblong 0.10 cm² 2 to 4 ml BS4 66-0042 4 x 8 mm Oblong 0.28 cm² 2 to 4 ml Specialty Vertical Chambers

BS4 66-0008 Snapwell^™ 1.13 cm² 4 to 6 ml BS4 66-0044 468.00 12mm Millicell^® 1.13 cm² 4 to 6 ml

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Tissue/Cell Culture

Horizontal Diffusion Chamber System

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 which occurs in the normal in vivo environment such as with nasal, pul- monary, corneal, or dermal cells.

The chambers create 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 System consists of six horizontal chambers, and six closed open chamber tops into which either Snapwell^™ devices or tissue mounting rings may be fitted on a horizontal plane, and a heat block. The closed perfusion system also accepts optional diffusion chamber electrodes for resistance measurements and electrophysiology. Either a peristaltic or syringe pump (not included with the system, see Section A in our Full-Line Bioscience Catalog) 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 the open or closed configuration.

• Designed for transport studies on cells and tissues which are exposed to an air interface — nasal, corneal, pul- monary or dermal

• Option of using cultured cells or tissue samples makes the horizontal diffusion chamber system versatile and convenient for correlation studies

• Six simultaneous tests can be run, improving experimental precision

• Heat block allows system temperature control with the use of a circulating water bath

• Option of working in open or closed configuration

• Compact configuration 38.1 x 8.9 x 10.2 cm (15 x 3.5 x 4 in) conserves valuable bench space

References:

1) G.M. Grass and S.A. Sweetana, “In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell,” Pharm. Res., 5372-76 (1988).

2) I.J. Hidalgo, K.M. Hilgren, G.M. Grass and R.T. Borchardt, “Characterization of the unstirred water layer in Caco-2 cell monolayers using a novel diffusion apparatus,”

Pharm. Res., 8:222-227 (1991).

For additional references, please visit our website.

Vertical System Accessories

BS4 66-0021 24 Chamber Heating Block for Vertical System

Catalog No. $ Product

Accessories and Electrodes

BS4 66-0007 55.00 Retaining Ring and O-Ring, pkg. of 12 BS4 66-0009 1,196.00 6-Chamber Heating Block

BS4 66-0010 414.00 Clear Front Panel BS4 66-0012 43.00 Retaining Ring Clamp Tool

BS4 66-0018 93.00 Tissue Chamber Electrode Cap, pair L/R BS4 66-0019 93.00 Tissue Chamber Perfusion Cap, pkg. of 2 BS4 66-0020 93.00 Electrode Cap for Snapwell^™Chambers, pair L/R BS4 66-0021 1,720.00 24-Chamber Heating Block

BS4 66-0025 1,256.00 24 Leads Voltage/Current Electrode Manifold, 2mm pin sockets

BS4 66-0048 1,113.00 12 Channel Air Manifold

BS4 66-0050 12.00 Electrode Cap Screws for BS4 66-0018 and BS4 66-0020, pkg. of 12

BS4 66-0023 443.00 Ag/AgCl Electrodes with Potassium Chloride, pkg. of 4, with O-rings

BS4 66-0024 131.00 Glass Barrel for Electrode with Ceramic Tip, pkg. of 8 BS4 66-0049 100.00 Open Screws (with step) and O-rings, serves as

Electrode Holders for Vertical Low Volume Diffusion Chambers, pkg. of 12. This step will allow the electrode to be positioned in the same place every time BS4 66-0057 47.00 Small O-rings for Electrodes, pkg. of 24

BS4 66-0006 355.00 Replacement Air Manifold Line for BS4 66-0011 only, 6 inch long, pkg. of 12

BS4 7507-023 33.00 Replacement Air Manifold Line for BS4 66-0011 only, 6 inch long, 1 ea.

BS4 7507-028 47.00 Replacement Air Manifold Line for BS4 66-0011 only, 36 inch long, 1 ea.

For Thermocirculators, see pages O2 to O7.

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Harvard Apparatus has a wide range of circulating water baths which can be used to maintain the heat block temperature, see pages O2 to O7

Horizontal Diffusion Chamber System

We offer a single horizontal diffusion chamber and a 6-chamber system. The system includes the following components:

Catalog No. Qty. Product

BS4 66-0017 1 Heat Block for Horizontal Chambers, for 1 to 6 Chambers BS4 66-0016 6 Horizontal Diffusion Chambers Horizontal Diffusion Chamber Horizontal Diffusion Chamber Cross Section

Open Configuration

The upper (apical) surface of the cells is directly accessible, making the system suitable for drug transport or cytotoxicity testing of liquids and semi-solid materials.

Closed Configuration

Cells or tissue can be exposed to solutions, perfused with gas or liq- uid, or gas pressure can be applied. The closed perfusion system also accepts Diffusion Chamber Electrodes for resistance measurements and electrophysiology.

Diffusion Chamber Electrodes

Diffusion Chamber Electrodes, see diagram on page 191, can be used in both Vertical and Horizontal Diffusion Chambers for making monolayer resistance measurements and electrophysiological determinations. The 2 mm pin connections are compatible with our electrical manifolds that connect the electrodes to various models of Current/Voltage Clamps, see pages M176 to M182. The silver/silver chloride reference electrode consists of a chlorided silver wire contained in a glass barrel that terminates in a ceramic tip. The barrel is filled with any suitable electrolyte solution, usually the buffer used for the external media or 3M KCl, and is refillable. The microporous ceramic tip limits the flux of ions into and out of the electrodes, making them a suitable replacement for bridge systems in most applications. The electrode connects to a wire 1 m in length and is terminat- ed with a 2 mm gold plated pin.

Horizontal Diffusion Chamber System Catalog No. $ Chamber Included Horizontal Diffusion Chambers System

BS4 66-0005 Horizontal Diffusion System with 6 Horizontal Chambers

BS4 66-0016 737.00 Horizontal Diffusion Chamber with Open and Closed Caps

Electrodes and Accessories

BS4 66-0017 1,232.00 Heat Block for Horizontal Chambers, for 1 to 6 Chambers

BS4 66-0022 Tissue Mounting Ring for Horizontal Chamber Only BS4 66-0053 25.00 Closed Screws, used when perfusion is not need-

ed, pkg. of 24

BS4 66-0054 Closed Caps for Horizontal Chambers BS4 66-0055 78.00 Open Caps for Horizontal Chambers BS4 66-0056 Male Luer Fitting - 3/32 Bar, pkg. of 24 BS4 66-0023 448.00 Ag/AgCl Electrodes with Potassium Chloride,

pkg. of 4

BS4 66-0024 Glass Barrel for Electrode with Ceramic Tip, pkg. of 8

BS4 66-0057 47.00 Small O-rings for Electrodes, pkg. of 24 BS4 66-0058 Open Screw (straight hole) and O-rings, serves as

an electrode holder for Horizontal Diffusion Chambers, pkg. of 24

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Tissue/Cell Culture

Ussing Chambers

U9500 "Classic" Design Ussing Chamber System

The U9500 is a complete system consisting of Ussing chamber, support stand, circulation reservoir, ring stands, 3-prong clamp, air/gas fitting kit and electrode set.

U9500 Series Ussing Chambers

U9500 series Ussing chambers are machined from solid clear acrylic rod into two symmet- ric halves. Each half has locating holes on the outside end for mounting onto the support bracket. One face has sharp stainless-steel pins which mate with holes in the correspon- ding face for puncturing and positioning the epithelium membrane. Luer-tapered fittings are pre-installed in the vertical and horizontal ports for circulation of buffer solutions and for making electrical I/O connections into the chamber.

Choice of 5 Diameters and 3 Slotted Sizes

U9500 series Ussing chambers are available in five diameters from 3.8 mm to 13.5 mm and three slotted sizes to accommodate a variety of tissue sizes. The choice of chamber depends on the species and epithelia under study. The specified diameter designates the main chamber opening in which the buffer makes contact with the membrane.

U9521 Cell Culture Insert Ussing Chamber and Adapters

The use of permeable cell culture inserts (porous membrane filters, culture cups, and culture wells) has become widespread in recent years. The numerous advantages of this application have revolutionized in vitro cell biology and it is now used extensively in electrophysiological studies of epithelium.

The U9521 Ussing chamber is designed to hold tissue culture inserts using low-cost adapters. The chamber is mounted in the standard support stand and procedures for experimentation are the same as with the

"classic" Ussing system. This is a very versatile and economical system for using tissue culture inserts in transepithelial studies.

Adapters in a large variety of sizes for most brands of culture inserts are available.

U9500 Classic Ussing System (preamp not included)

The investigation of ionic transport across epithelial cells, whether by diffusion or active transport, was greatly facilitated by the development of research equipment specifically for this purpose by Dr. H. H. Ussing more than 40 years ago. Today this equipment bears his name.

The “classic” design presented here varies little from the original equipment developed by Dr. Ussing and this tried-and-true system is still the most widely used equipment in the study of electrophysiological properties of epithelial tissues, particularly the investigation of potential difference (PD), resistance (R), and short-circuit current (Isc). A newer design has recently been introduced allowing the insertion of cell culture supports into the Ussing chamber greatly increasing its functionality. The low initial cost of this equip- ment, coupled with the modest cost of add-on components and longevity of service, make the "classic" Ussing system ideal for classroom instruction and in-the-field research.

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Ussing Chambers

Circulation Reservoirs, 4 Volumes

The circulation reservoir is hand-blown from borosilicate glass and is available in four fluid capacities. Two glass condensers are included to minimize evaporative losses.

Reservoir openings are provided to allow the introduction of compounds at any time during the experiment. In addition, there are three sets of openings for connections.

Each side of the reservoir has two outlets to which a short piece of tubing connects to the fittings in the vertical ports of the Ussing chamber. This is the buffer circulation connection.

Each side of the reservoir has one inlet for an air/gas connection. Gas, commonly a 95% O₂/5% CO₂ mixture, is forced under low pressure into these openings and allowed to come into contact with the buffer solution. Bubbles, thus formed, generate a

Classic Tissue Chamber

Circulation Reservoir U9521 Culture Cup

Ussing Chamber

circulation and oxygenate the buffer. This is commonly referred to as a gas-lift perfusion system.

The center of the reservoir contains one inlet and one outlet for water circulation.

Water is pumped through the water jackets which surround the buffer circulation system and maintains the perfusion solutions at constant temperature. Using this system, the effect of temperature on the subject epithelium can be studied.

Support Stand

The support stand is manufactured from white acrylic and is mounted on four rubber, no-slide feet. The support stand includes anodized alu- minum hardware comprised of the pre-mounted chamber support bracket, two threaded ring stands and one 3-prong utility clamp.

U9975A Electrode Set

Each chamber is supplied with a a set of 4 electrodes, 2 Ag-AgCl pellet electrodes for voltage and 2 Ag wire electrodes for current. The electrodes include 1 meter (3 feet) of wire and a connector to plug into the amplifier headstage.

Bridge Fittings

Luer taper fittings screw onto the electrodes to provide agar salt bridge connection to the chamber. The fittings are filled with agar either before or after assembly to the electrode, depending on the length and tip size of the fitting.

*All dimensions in millimeters

Electrodes and Bridge Fittings*

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Ussing Chambers

Catalog No. $ Model Product

When ordering Complete System, please specify Chamber Model and Circulation Reservoir size.

U9500 Classic Ussing Systems

Each System includes 1 Chamber, Support Stand with 2 Ring Stand Rods, Circulation Reservoir, 3-Prong Clamp, Air/Gas Fitting Kit U9303, Electrode Set U9975A and Electrode Bridge Fitting Kit U9965EP. Select Chamber and Circulation Reservoir from list below. Systems with suffix DR include Drain/Buffer Ports with Stopcock Valves.

BS4 64-0500 852.00 U9500 Classic Ussing System with Round Chamber

BS4 64-0501 911.00 U9500DR Classic System with Round Chamber and Drains

BS4 64-0502 911.00 U9500S Classic Ussing System with Slotted Chamber BS4 64-0503 968.00 U9500SDR Classic System with Slotted

Chamber and Drains

Note: U9500 System components ordered separately.

Chambers

BS4 64-0504 204.00 U9504 Ussing Chamber, 3.8 mm D BS4 64-0505 204.00 U9506 Ussing Chamber, 6.0 mm D BS4 64-0506 204.00 U9509 Ussing Chamber, 9.0 mm D BS4 64-0507 204.00 U9512 Ussing Chamber, 12.0 mm D BS4 64-0508 204.00 U9513 Ussing Chamber, 13.5 mm D BS4 64-0509 250.00 U9504SP-1 Slotted Chamber, 1.5 x 7.0 mm BS4 64-0510 250.00 U9504SP-2 Slotted Chamber, 2.5 x 7.0 mm BS4 64-0511 250.00 U9504SP-3 Slotted Chamber, 3.5 x 7.0 mm

*Note: Chambers with drains are available. Please call our technical support for more information. .

Electrode Components

BS4 64-0553 54.00 U9565EP Electrode Bridge Fittings, pkg. of 12, and Adapters, pkg. of 6

BS4 64-0554 146.00 U9975A Electrode Set, 2 Ag/AgCl Pellet, 2 Ag Wire

BS4 64-0555 27.00 U9985 Bridge Fitting Adapters, pkg. of 6 Circulation Components

BS4 64-0556 227.00 U9304 Circulation Reservoir, 4 ml/side BS4 64-0557 227.00 U9310 Circulation Reservoir, 10 ml/side BS4 64-0558 227.00 U9320 Circulation Reservoir, 20 ml/side BS4 64-0559 227.00 U9330 Circulation Reservoir, 30 ml/side BS4 64-0560 38.00 U9302 Condenser, set of 2, all sizes Additional Components

BS4 64-0561 15.00 U9303 Air/Gas Connection Kit

BS4 64-0562 185.00 U9403 Support Stand with One Ring Stand Rod

BS4 64-0563 37.00 U9403R Ring Stand Rod BS4 64-0564 41.00 U9404 3-Prong Clamp

BS4 64-0567 35.00 SL-6U Stopcocks with Luer Connectors for U9926 and U2500

BS4 64-0165 36.00 SL-6 Stopcocks, pkg. of 6

U9520 Classic Cell Culture Ussing Systems

Each system includes U9520 or U9520DR Chamber with Insert, Support Stand with 2 Ring Stand Rods, Circulation Reservoir, 3-Prong Clamp, Air/Gas Fitting Kit U9303, Electrode Set U9975A and Electrode Bridge Fitting Kit U9965EP. Select Chamber/

Insert Combination from below. See list opposite for Circulation Reservoirs. Systems with suffix DR include Drain/Buffer Ports with Stopcock Valves.

BS4 64-0520 968.00 U9520/A System with O-Ring Insert BS4 64-0521 1,003.00 U9520/B System with Round Insert

with Pins

BS4 64-0522 1,061.00 U9520/F System with Falcon^®Adapter BS4 64-0523 985.00 U9520/M System with Millicell^®Adapter BS4 64-0524 985.00 U9520/S System with Snapwell^™Adapter BS4 64-0525 1,061.00 U9520/T System with Transwell^®Adapter

Note: U9500 System components ordered separately.

BS4 64-0532 243.00 U9521 Cell Culture Ussing Chamber BS4 64-0533 301.00 U9521DR Cell Culture Chamber with Drains Tissue Adapters

BS4 64-0534 126.00 U9524A-04 3.8 mm Round Insert with O-Ring BS4 64-0535 126.00 U9524A-06 6.0 mm Round Insert with O-Ring BS4 64-0536 126.00 U9524A-09 9.0 mm Round Insert with O-Ring BS4 64-0537 126.00 U9524A-12 12.0 mm Round Insert with O-Ring BS4 64-0538 126.00 U9524A-12 13.5 mm Round Insert with O-Ring BS4 64-0539 132.00 U9524B-04 3.8 mm Round Insert with 5 Pins BS4 64-0540 132.00 U9524B-06 6.0 mm Round Insert with 5 Pins BS4 64-0541 132.00 U9524B-09 9.0 mm Round Insert with 5 Pins BS4 64-0542 132.00 U9524B-12 12.0 mm Round Insert with 5 Pins BS4 64-0543 132.00 U9524B-13 13.5 mm Round Insert with 5 Pins Culture Cup Adapters

BS4 64-0544 193.00 U9524F-09 Falcon^®Adapter, 9 mm BS4 64-0545 193.00 U9524F-12 Falcon^®Adapter, 12 mm BS4 64-0546 193.00 U9524F-25 Falcon^®Adapter, 25 mm BS4 64-0547 132.00 U9524M-12 Millicell^®Adapter, 12 mm BS4 64-0548 132.00 U9524M-30 Millicell^®Adapter, 30 mm BS4 64-0549 132.00 U9524S Snapwell^™Adapter BS4 64-0550 193.00 U9524T-06 Transwell^®Adapter, 6.5 mm BS4 64-0551 193.00 U9524T-12 Transwell^®Adapter, 12 mm BS4 64-0552 193.00 U9524T-24 Transwell^®Adapter 24 mm

See left column for electrode and additional components.

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Tissue/Cell Culture

Self-Contained Ussing Chambers U2500 and U9926

U9926 Single Channel Chamber U2500 Dual Channel

Chamber (2 Inserts)

U2500 Dual Channel Chamber (2 Inserts) Exploded View

The self contained chambers are offered in single and dual channels. Warner self-contained Ussing chambers offer many advantages.

Solution warming is provided by pumping heated water through the chamber halves. There are no delicate glass heat exchangers to be broken.

The chamber consists of two holder halves which clamp around the chamber insert. Each half has 4 ports into the bath compartment, 2 electrode ports for the voltage and current electrodes, an air/gas port and a drain port.

Inserts are available for a variety of tissue sizes and for the many culture cups commonly used. Standard tissue sizes are shown in the chart on the following page.

Inserts are quickly changed between experiments.

Solutions are drained from each chamber half through drain valves provided. The halves are then unclamped and the insert is removed and replaced with the new one.

The dual chamber U2500 has an additional feature.

It includes 4 needle valves for individual adjustment of the air/gas mixture into each of the chamber halves.

• No glass circulation reservoirs

• Modular design

• Fast change Plug-in Cartridge (A)

Plug-in Cartridge (B)

Chamber Half-Cell Reservoir Cover

Ag Wire (Current Electrode)

Electrode Bridge Fittings Bridge Fitting Adapters Drain

Valves

Ag/AgCl Pellet (Voltage Electrode)

Clamp Screws

Upright Support, Air/Gas Manifold Needle Valves

Base Plate Air/Gas Luer Connectors

O-Ring Seals Water Jacket

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Self-Contained Ussing Chambers U2500 and U9926

Insert Selection for U9926 & U2500 Choose Inserts from Following List:

Tissue Inserts

BS4 64-0582 105.00 U9924A-04 Round Insert, 3.8 mm, with O-Ring BS4 64-0583 105.00 U9924A-06 Round Insert, 6.0 mm, with O-Ring BS4 64-0584 105.00 U9924A-09 Round Insert, 9.0 mm, with O-Ring BS4 64-0585 105.00 U9924A-12 Round Insert, 12.0 mm, with O-Ring BS4 64-0586 105.00 U9924A-13 Round Insert, 13.5 mm, with O-Ring BS4 64-0587 138.00 U9924B-04 Round Insert, 3.8 mm, with 5 Pins BS4 64-0588 138.00 U9924B-06 Round Insert, 6.0 mm, with 5 Pins BS4 64-0589 138.00 U9924B-09 Round Insert, 9.0 mm, with 5 Pins BS4 64-0590 138.00 U9924B-12 Round Insert, 12.0 mm, with 5 Pins BS4 64-0591 138.00 U9924B-13 Round Insert, 13.5 mm, with 5 Pins BS4 64-0592 165.00 U9924C-03 Slotted Insert, 1.6 x 7.0 mm, with

6 Pins

BS4 64-0593 165.00 U9924C-04 Slotted Insert, 2.4 x 7.0 mm, with 6 Pins

BS4 64-0594 165.00 U9924C-05 Slotted Insert, 3.5 x 7.0 mm, with 6 Pins

Culture Cup Inserts

BS4 64-0595 193.00 U9924F-09 Falcon^®Adapter, 9 mm BS4 64-0596 193.00 U9924F-12 Falcon^®Adapter, 12 mm BS4 64-0597 193.00 U9924F-25 Falcon^®Adapter, 25 mm BS4 64-0598 132.00 U9924M-12 Millicell^®Adapter, 12 mm BS4 64-0599 132.00 U9924M-30 Millicell^®Adapter, 30 mm BS4 64-0600 132.00 U9924S Snapwell^™Adapter, 12mm BS4 64-0601 193.00 U9924T-06 Transwell^®Adapter, 6.5 mm BS4 64-0602 193.00 U9924T-12 Transwell^®Adapter, 12 mm BS4 64-0603 193.00 U9924T-24 Transwell^®Adapter, 24 mm Additional/Replacement Parts

BS4 64-0565 71.00 U9406 Tubing/Fitting Kit; Includes 3/16 in x 10 ft (ID x L) Tygon Tubing, 1/16 in x 10 ft (ID x L) Tygon Tubing, Eighteen 1/16 in Fittings (six each of straight, Y and Luer Fittings) BS4 64-0553 54.00 U9565EP Electrode Bridge Fitting Kit; Includes

twelve Bridge Fittings, six Bridge Fitting Adapters

BS4 64-0554 146.00 U9975A Electrode Set; Includes two Ag-AgCl Pellet Voltage Electrode, two Ag Wire Current Electrode BS4 64-0555 27.00 U9985 Bridge Fitting Adapters, pkg. of 6

Note: Prices are for replacement or additional units ordered.

U9926 Single Channel Self-Contained Ussing Chamber

Each U9926 is supplied with (1) Insert. Model number suffix indicates type of Insert. Tissue Types include (A) Round with O-Ring, (B) Round with Sharp Pins and (C) Slotted with Pins. Culture Types include (F) Falcon, (M) Millicell, (S) Snapwell and (T) Transwell. Each type Insert is supplied in various sizes. Consult adjoining table to select size and include information with your order.

Standard Accessories Supplied with U9926: 1 Electrode Set (2 voltage/2 cur- rent) p/n U9975A; 12 Bridge Fittings with 6 Adapters p/n U9565EP; 1 Tubing/

Fitting Kit p/n U9406

BS4 64-0568 1,621.00 U9926/A U9926 with (A) O-Ring Insert BS4 64-0569 1,653.00 U9926/B U9926 with (B) Round/Pin Insert BS4 64-0570 1,675.00 U9926/C U9926 with (C) Slotted/Pin Insert BS4 64-0571 1,709.00 U9926/F U9926 with (F) Falcon^®Insert BS4 64-0572 1,638.00 U9926/M U9926 with (M) Millicell^®Insert BS4 64-0573 1,638.00 U9926/S U9926 with (S) Snapwell^™Insert BS4 64-0574 1,709.00 U9926/T U9926 with (T) Transwell^®Insert U2500 Dual Channel Self-Contained Ussing Chamber

Each U2500 ordered is supplied with (2) Inserts. Model number suffix indicates type of Insert. Tissue types include (A) Round with O-Ring, (B) Round with Sharp Pins and (C) Slotted with Pins. Culture types include (F) Falcon, (M) Millicell, (S) Snapwell and (T) Transwell. Each type Insert is supplied in various sizes. Consult adjoining table to select size and include information with your order.

Standard Accessories Supplied with U2500: 2 Electrode Sets (2 voltage/

2 current) p/n U9975A; 2 Sets of 12 Bridge Fittings with 6 Adapters p/n U9565EP;

2 Tubing/Fitting Kits p/n U9406

BS4 64-0575 2,719.00 U2500/A U2500 with (A) O-Ring Insert BS4 64-0576 2,751.00 U2500/B U2500 with (B) Round/Pin Inserts BS4 64-0577 2,774.00 U2500/C U2500 with (C) Slotted/Pin Inserts BS4 64-0578 2,811.00 U2500/F U2500 with (F) Falcon^®Inserts BS4 64-0579 2,735.00 U2500/M U2500 with (M) Millicell^®Inserts BS4 64-0580 2,735.00 U2500/S U2500 with (S) Snapwell^™Inserts BS4 64-0581 2,811.00 U2500/T U2500 with (T) Transwell^®Inserts

Tissue Insert Sizes, Openings and Pin Layouts Tissue Insert Sizes, Openings and Pin Layouts

*All dimensions in millimeters

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Tissue/Cell Culture

Voltage/Current Clamps

• Provides control (clamping) of either voltage or current across tissues or cultured cell monolayers

• Fully compatible with EasyMount, Harvard NaviCyte and most other lines of diffusion/Ussing chamber systems

• Suitable for both electrophysiological and ion flux studies

• Available in a variety of configurations

• Optional high voltage output

• Digital readout for displaying voltage and current

• Electrode offset voltage compensation

• User selectable gains for

transepithelial current measurements

• Pulse generator for continuous monitoring of the tissue conductance

• Fluid resistance compensation with user selectable ranges

• Master control feature

• Versatile electrode input modules/

panel for easy electrode connections

• Computer interface connection for data acquisition and control

The short-circuit technique introduced by Ussing and Zerhbn* has been and will continue to be, an extremely valuable methodology to study mechanisms and regulation of epithelial transport. The technique has been instrumental toward understanding the basic physiology of solute and water transport by epithelia, pathophysiological changes such as secretion induced by pathogens like cholera, genetically altered transport as in cystic fibrosis, pharmacological modulation of transport as by important agents like diuretics and inhibitors of acid secretion, and homeostatic mechanisms underscored by the epithelium’s role as a selective barrier that separates and maintains the pristine internal environment from the harsh, external world.

Essential to this methodology is instrumentation suitable to the wide variety of epithelial types being studied which range from isolated, perfused renal tubules to small biopsy specimens, from primary cell cultures to established cell lines, from low resistance, leaky tissues like small intestine to very tight epithelia such as urinary bladder. The VCC series of voltage/cur- rent clamps have been designed to meet these tissue requirements. The VCC instruments have proven to be flexible and versatile laboratory tools for studying ion transport mechanisms in a wide range of epithelia. These instruments are available in both benchtop and rack mountable models from the VCC600 single channel system to our latest multichannel model, the VCC MC8, which can be configured with up to 8 individual clamp modules.

Flexibility has been built into all VCC series voltage and current clamps such as user-selec- table frequency response, current gain and fluid resistance compensation ranges that enable the investigator to optimize the instrument for use on a wide variety of tissue types and chamber configurations which vary from those for very small tissue samples such as biopsy specimens (e.g., 0.01 cm²) to larger tissue specimens or cell cultures (e.g., > 1 cm²).

A built-in pulse generator enables automatic generation of unipolar or bipolar pulses for determining transepithelial resistance. Outputs are provided for continuous recording of transepithelial current and voltage. A remote control interface permits any of the VCC

family of instruments to be controlled by an external device such as a computer. * Ussing, H.H., Zerhbn, K. 1951. Active transport of sodium as the source of electric current in the short circuited isolated frog skin. Acta Physiol. Scand. 23:110

BS4 69-0938 6-Channel Voltage/Current Clamp shown with Harvard NaviCyte Vertical Diffusion Chamber System, see page M166

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Tissue/Cell Culture

Epithelial Voltage Clamps

Single Channel EC-800 &

EC-800LV

Epithelial voltage clamps from Warner Instrument Corp. provide accurate measurements of transepithelial voltage, short circuit current and membrane resistance. Important features include fluid resistance compensation, membrane resistance readout, choice of voltage compliance and small water-tight preamp head- stages. Operating modes include Voltage Clamp, Current Clamp, Voltmeter and Resistance.

Unique Features

Epithelial Voltage Clamps from Warner are state-of-the-art instruments with several unique and important design features offering more reliable recording and operator convenience.

Membrane Resistance Measurement

Accurate resistance measurements are made with the membrane mounted in the chamber.

The measurement is made using a low frequency [2 Hz] bipolar signal to avoid polar- ization of the membrane [ideal for monolayers]. Resistances up to 200 kΩ are displayed on the meter with push-button convenience.

Clamp Speed Selection:

10 µsec Clamping

Three clamp speeds provide optimum recording conditions for a variety of applications. In Fast mode, preparations with low access resistance (small tissues or monolayers) can be clamped with speeds as fast as 10 µsec.

Typical Ussing chambers with larger tissues will use Medium or Slow modes for stable clamping free from oscillations.

Independent Voltage and Current Commands

Internal DC Command (Hold) controls are provided for both Voltage clamp and Current clamp modes.

Watertight Headstage with Model Membrane

The small compact headstage can be located close to the measurement site to keep input leads short for reduced noise pick-up. The model membrane circuit simulates a prepara- tion to provide convenient operational checks of the clamp. Internal circuits are protected against the invasion of corrosive saline solu-

Choice of Voltage Compliance

Models EC-800: ±120 V

The high voltage compliance model EC-800 is important for studies of low resistance [leaky]

epithelial cells and in applications in which long agar leads in the current passing circuit produce large voltage drops which must be compensated. Additionally, high compliance helps in charging large membrane capaci- tances typical of epithelial tissues, resulting in faster settling times and improved overall clamp performance.

Models EC-800LV: ±15 V

Studies with small tissue samples or monolayers in set-ups with low access resistance may not require high compliance. For this application, model EC-800LV offers both a lower cost and a safer environment for the membrane.

Additional Features

• High CMR: Differential voltage recordings are made with very high common mode rejection providing accurate measurements free from the effects of common mode potential changes of a noisy environment.

External Control

The clamp can be operated by an external programmer, lab timer or computer. Logic control of clamp mode and clamp command levels is possible as well as simultaneous mixing of external linear commands.

• For studies of epithelial transport and the electrical properties of tissue

• Membrane Resistance Measurement Circuit

• Clamp Speed Selection

• Independent Voltage and Current Commands

• Watertight Headstage with Model Membrane

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Tissue/Cell Culture

Specifications

EC-800 & EC-800LV Headstage:

Input Impedance 1 x 10¹⁰Ω shunted by 6 pF Input Voltage ±1.5 V max.

Common Mode Voltage ±13 V max.

Common Mode Rejection 100 dB @ 60 Hz Leakage Current 20 pA max.

Offset Voltage Range ±120 mV Voltage Clamp Ranges:

Int. Clamp Potentiometer ±100 mV with 10-turn control

Ext. Command ±1 V

Ext. Command Factor 1 mV/10 mV applied Current Clamp Ranges:

Clamp Potentiometer ±1 mA External Command ±10 mA Command Factor 1 µ A/10 mV applied

Speed 10 µ sec measured with model membrane Resistance:

Fluid Resistance 0-100 Ω standard Compensation Range 0-1 kΩ optional Membrane Resistance

Measurement (made with 2 Hz bipolar constant current square wave)

Ranges 0-2 kΩ, injected current = 10 nA 0-200 kΩ, injected current = 1 nA Panel Meter 3-1/2 digit LCD; Voltage Range: 200 mV max;

Current Range: 2000 µ A max Outputs:

Voltage Monitor x10

Current Monitor 10 mV/µ A Compliance:

EC-800 ±120 V

EC-800LV ±15 V

Power Requirements 100-130 VAC or 220-240 VAC, 50/60 Hz, 15 VA Physical Dimensions, H x W x D:

EC-800 & EC-800LV 8.9 x 43.2 x 30.5 cm

Headstage 7.7 x 7.7 x 5 cm

Shipping Weight 6.4 kg

Warranty Three years, parts & labor

BS4 64-0035 2,619.00 EC-800 Single Channel Voltage Clamp with

±120 V Compliance*

BS4 64-0036 2,310.00 EC-800LV Single Channel Voltage Clamp with

±15 V Compliance*

*Supplied with rack mount hardware. Specify line operating voltage if not 100-130 VAC.

Epithelial Voltage Clamps

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