MyoBath INSTRUCTION MANUAL World Precision Instruments An organ bath system for the study of isolated tissues in vitro Serial No.

Transcription

1 An organ bath system for the study of isolated tissues in vitro INSTRUCTION MANUAL Serial No. Developed In collaboration with McPherson Scientifi c Pty., Ltd World Precision Instruments

2 Contents Introduction... 1 System Composition... 2 List of Parts... 3 System Assembly... 4 Step 1: Installation of leveling feet and waste disposal adapters... 4 Step 2: Installation of waste manifold, stainless steel rods (and red caps).. 6 Step 3: Installation of micrometer brackets... 7 Step 4: Installation of the stimulation manifold and aeration manifold... 7 Step 5: Installation of organ baths... 8 Step 6: Installation of force transducer... 8 Step 7. Installation of tissue holder... 8 Step 8: Connection of tubing... 8 General Experimental Protocol Experimental Procedures Additional Information on Holders and Tissue-mounting Warranty Copyright 2001 by World Precision Instruments, Inc. All rights reserved. No part of this publication may be reproduced or translated into any language, in any form, without prior written permission of World Precision Instruments, Inc. WORLD PRECISION INSTRUMENTS 18

3 Introduction The use of isolated tissues in the study of physiological and pharmacological function of organs has been performed for most of this century. To a large extent it is the mechanical properties of isolated tissues that interests us the most, however the underlying electrical, biochemical (second messengers) and ionic (ion fluxes) alterations that mediate these responses are obviously important aspects of organ function. To a large extent these properties can only be classifi ed when specifi c tissues are studied in isolation. In terms of in vitro techniques, characterization of many of the properties of isolated tissues (such as blood vessels) can occur at a number of different levels of organ integrity. WPI s multichannel organ bath system is designed through years of research and development for simultaneous studies of isolated tissues in vitro. This easy-to-use, cost-effective bench top system is suitable for experiments on vascular and non-vascular smooth muscle and cardiac muscle. Coronary artery, taenia coli, isolated atria and ventricle strips have all been successfully studied using the organ bath. System Not shown: Gravity feed assembly for warming tissue buffer solution. WORLD PRECISION INSTRUMENTS 1

4 Composition The basic system uses vertical stainless steel rods mounted on a sturdy PVC base. The system also includes an aeration manifold which controls distribution of carbogen to the organ baths, tissue preparation dish and buffer reservoir; a stimulation manifold which also forms the top cross support; and a waste manifold which is mounted on the back side of the base plate. The organ baths and buffer reservoir are jacketed and a heating pump (such as the Haake DC1-B3 circulating bath, available from WPI) is needed to maintain the buffer at a desired temperature. WPI currently supplies three sizes of organ bath (10, 25 and 50 ml) and a one-liter buffer reservoir. The heart of each individual channel is the bracket made of two PVC blocks. One of the block (smaller one) is used to install the strain gauge FORT 10 or FORT 100 (full scale 10 or 100 grams) for isometric study of isolated tissues. A micrometer is mounted on the other block to position an attached tissue holder. The micrometer not only improves the ease of positioning the tissue preparation into or out of the bath, but also provides users with a useful tool in the study of such tissues as vascular segments where accurate diameter-tension curves can be constructed in order to optimize the passive force placed on the tissue (McPherson, 1992). Currently, there are six types of tissue holders available, atria holder with stimulation feature (AS), vascular holder (V), vascular holder with stimulation (VS), blank holder with stimulation (B), microtissue holder (MH1), and microtissue holder with stimulation feature (MHS1). WORLD PRECISION INSTRUMENTS 2

5 List of Parts (for four-channel organ bath) Quantity Base... 1 (including base plate and stimulation cross support) Stainless steel rod... 4 Aeration (carbogen) manifold... 1 Waste manifold... 1 Micrometer bracket... 4 Tissue holders (choice of four types: AS, V, VS, B)... 4 Organ bath (10, 25 or 50 ml)... 4 Organ bath extension clamp... 4 Closed clamp... 4 Force transducers (FORT10 or FORT100) channel Transbridge amplifi er (TBM4M)... 1 Start-up kit*... 1 * Start-up kit includes silicone, nylon and vinyl tubing; twelve fl athead screws (8x25 mm, 2x30 mm and 2x40 mm in length); six leveling mount feet with studs; eight black NPT male pipe adapters (4 straight and 4 right-angled); fi ve vessel clips; four aeration connection adapters (blue plastic); one circular level measure and one 5-gram scale weight. Additional accessories include Haake DC1-B3 circulating Bath, WPI s FORT 250 (full scale 250 grams) or FORT 1000 (full scale 1000 grams) force transducers, and eight channel Bridge-8 amplifi er available for applications where higher resolution of force-transduction is needed or more than four tissue preparations need to be studied simultaneously. Outputs from TBM4M or Bridge-8 amplifi ers can be fed into chart recorder(s) or collected and processed with WPI s CVMS data acquisition system. This data acquisition and analysis system is especially useful in the on-line analysis of contractile responses. WPI supplies a number of stimulators (A300, A310) and stimulus isolators (A360, A365, A385). Together with the timing programmer PRO-4, these constant current generators are ideally suited for use with the MyoBath system. WORLD PRECISION INSTRUMENTS 3

6 System Assembly The following procedure provides you with the information needed to assemble the organ bath system. Step 1: Installation of leveling feet and waste disposal adapters Screw one hex nut onto each of the six leveling feet. Then install the feet on the bottom side (i.e., the side without the grove) of the PVC base plate. Using the circular level provided, adjust the feet so that the base plate is in a fl at plane, then tighten the hex nuts up against the base plate. The eight black NPT male pipe adapters (4 straight and 4 right-angled) are provided to connect the drain from the organ baths to a waste manifold through silicone tubing underneath the plate. Screw the four straight pipe adapters into the (screw) holes (D) on the top of base plate, and the four right-angled adapters into the same holes from underneath. Make sure that barbed outlet of each rightangled adapter points toward the rear of the base where the waste manifold will be attached. G G M G D M G D M M D D WORLD PRECISION INSTRUMENTS 4

7 The 1 /4 (I.D.) silicone tubing is used to connect the drain of the baths to the waste manifold. Two segments of tubing are needed for the waste connection from each bath: one connects the bath drain to the top disposal pipe adapter on the base plate; the other connects the right-angled pipe adapter on the bottom of the base plate with the barbed connector on the waste manifold. Four tubing clamps are included to stop the fl ow from the drain during experiments. The Tygon tubing (1/2 O.D.) is used to connect the main outlet of waste manifold to an appropriate disposable site. WORLD PRECISION INSTRUMENTS 5

8 Step 2: Installation of waste manifold, stainless steel rods (and red caps) Use the two 40 mm fl athead screws to mount the waste manifold onto the back side of the base plate. Make sure that there is a rubber O-ring on the waste manifold, to ensure a fl uid-tight coupling between the manifold and the base plate. Insert the stainless steel rods into central row of four holes (marked M in the fi gure below) and use four of the eight 25 mm fl athead screws to secure them to the base plate from the bottom. Slide the closed clamp onto each stainless steel rod as shown in the fi gure below. The four additional holes (G) are designed for the installation of a gravity fed buffer warming system (optional). Buffer is warmed in effi cient heat-exchanging glass coils in the system, before fl owing down into baths. The buffer warming system not only provides excellent temperature and fl ow control of tissue samples but also keeps both the buffer (and heated water) in closed loops so that there is no contaminant or mess when transferring it. The buffer warming systems GF-4, GF-3, or GF-2 are recommended for use with the MyoBath-4, -3, or -2 systems. For users choosing not four red plastic caps prevent spilled fluid to have the gravity feeding buffer warming system, are provided for insertion into the four holes (G) to from dripping through the base plate when transferring fl uid with a syringe. G G M G M G M D M D D D WORLD PRECISION INSTRUMENTS 6

9 Step 3: Installation of micrometer brackets The bracket is the heart of each individual channel, consisting of two PVC blocks with micrometer and strain gauge mounting sites. Align the 0.5 -in. hole on the bracket with the stainless steel rod, push it down onto the rod to appropriate height, then secure it with the knob on the side of the strain gauge block. aeration manifold stimulation manifold Step 4: Installation of the stimulation manifold and aeration manifold Align the four holes on the stimulation manifold with the four stainless steel rods, making sure that the BNC connectors with the hex nuts face downward. Push the stimulation manifold down onto the shoulders on the rod ends and fasten them with four 25 mm fl athead screws. Use the two 30 mm fl athead screws to install the aeration manifold onto the stimulation manifold. Make sure that the air outlets of the valves on the aeration manifold are facing downward. WORLD PRECISION INSTRUMENTS 7

10 Step 5: Installation of organ baths Insert the organ bath extension clamp into the closed clamp previously installed on the stainless steel rod (page 6). Adjust the jaw opening size of the extension clamp in order to mount an organ bath (be sure that the inlets and outlets of each bath faces the backside of the setup). Adjust the height of the four organ baths (if necessary) using the knob on the closed clamp. Step 6: Installation of force transducer FORT100 or FORT10 force transducers have been fi tted with adapters for mounting on the micrometer bracket. Insert the transducer into the strain gauge block and tighten it with the knob on the top of the block. Step 7. Installation of tissue holder Loosen the knob on a tissue holder (AS, V, VS, B, MH1 or MHS1), insert the micrometer s non-rotating head into the 1 /2 hole on the holder and tighten it up (be sure that the tissue mounting side of the holder is facing the front side of the MyoBath setup). Adjust the height and depth of the bath clamp and/or closed clamp so that the tissue mounting site is just above the organ bath. Step 8: Connection of tubing Use the 1 /4 (I.D.) silicone tubing to connect the inlets and outlets of the heated water jacket on the organ bath (Notice that the inlets are close to the bottom of the baths and outlets are close to the top). If more than one channel of organ bath are in the system, the baths should be interconnected sequentially, i.e., the outlet of one bath is connected to the inlet of the next bath. Heated water is supplied from a circulating pump such as WPI s Haake DC1-B3 model. WORLD PRECISION INSTRUMENTS 8

11 Nylon tubing ( 1 /4-in. O.D.) is provided to distribute carbogen from the aeration manifold to each organ bath, a tissue-dissecting dish and a buffer reservoir (1 liter, available from WPI). Measure a segment of tubing long enough to be connected between a miniature valve on the manifold and the aeration inlet of a bath. Notice that the aeration inlet in each bath has a frit (a porous plug) for bubbling the carbogen. To connect to the miniature valve on aeration manifold, push the nylon tubing fi rmly into the valve. To remove tubing from the valve, push in on the collar of the valve while pulling the tubing out. Use the aeration adapter (blue plastic) provided to connect the other end of the nylon tubing to organ bath. silicone tubing heated water outlet overflow to oxygen manifold heated water inlet drain oxygen aeration inlet aeration adapter silicone tubing WORLD PRECISION INSTRUMENTS 9

12 General Experimental Protocol The following sections deal with specifi c experimental procedures that have been used in conjunction with the MyoBath system as an aid to users obtaining the most productive use of the system. Of course, you will need a tissue source; these are discussed later. There are, however, several other items that you will need: Slide up plastic segments to establish approximate length of tissue hook. Attach clip under the segment, so clip rests on force transducer blade. 1. Buffer. The tissue must be bathed in a physiological salt solution. A Krebs-based solution commonly used with the MyoBath system is (composition in mm) NaCl 119, KCl 4.7, MgSO4.7H2O 1.17, KH2PO4 1.18, CaCl2 2.5, NaHCO3 25, and glucose 11 gassed with 5% CO2 in O2 (carbogen). 2. O2 supply. Buffer aeration and is commonly achieved using a mixture of 5% CO2 in O2 (referred to as carbogen). In this Krebs-based solution, the CO2 forms part of the buffer system and must be included. The ph of the resultant solution, after aeration, is approximately The carbogen mixture should be attached, through high pressure tubing, to the aeration manifold for distribution to individual organ baths and the buffer reservoir. Use the fl ow regulators, located on the upper MyoBath support, to regulate fl ow to the individual baths and to the buffer reservoir. The remaining line is usually employed to aerate the dissecting dish. transducer blade clip plastic segments tissue hook 3. Prepare tissue hooks. It is necessary to set the length of the tissue hook based on the length of your preparation. Wind the micrometer on each bath so that the tissue holder is at its lowest point. Adjust the length of the upper hook so that the preparation can be mounted easily and that suffi cient tension can be applied to the preparation once it is mounted. When using the punctate stimulating electrodes for atrial work, make sure the atria is positioned over the electrodes. For coarse adjustment of the effi cient length of the upper tissue hook, use the clips provided. To do this, hold the hook between its plastic segments with a clip and then set the clip on to the surface of force transducer. 4. Temperature regulation. The MyoBath system is jacketed and requires an external pumping system to keep the organ bath and buffer reservoir at the selected temperature. Normally the temperature is set at 37 o C although in some cases this has to be dropped to 33 o C to reduce the occurrence of spontaneous myogenic tone. There can be a signifi cant drop in the temperature of the recirculating solution around the system. It is wise therefore to check that the temperature of buffer-fi lled organ baths are at the required temperature rather than WORLD PRECISION INSTRUMENTS 10

13 assuming the temperature is what is set on the recirculating heater pump. 5. Transducer calibration. Calibrate the system before use so that the output you obtain is in meaningful units (e.g., grams force). Subsequently, the system calibration only needs to be rechecked to make sure these calibration values have been held. To calibrate the transducer, fi rst zero the output from the bridge amplifi er. Set your chart recorder to zero output. Then apply a 5 gram weight. Using the chart recorder s variable scaling, adjust the output so that the 5 gram weight gives the desired pen defl ection. (Users of CVMS data acquisition system should follow the calibration techniques described in the CVMS manual. Experimental Procedures 1. Preparing the MyoBath. Before you commence an experiment you should prepare the MyoBath to receive the tissues. First, all the electronics need to be turned on and allowed to warm for 15 minutes. The heater and circulator pump should be turned on. The carbogen supply should then be turned on and the baths and the buffer reservoir fi lled with buffer. (Note: Do not have buffer present in the baths/buffer reservoir without the carbogen supply on since buffer will drain back into the aeration tubing.) Wind the micrometer on each bath so that the lower tissue holder is fully elevated. After the system has had time to warm and equilibrate, calibrate the transducers. 2. Isolation and mounting of blood vessels. The following section describes a typical experimental protocol that involves setting up a pig coronary artery for study within the MyoBath. Porcine hearts from freshly killed pigs were obtained at a local abattoir. The right circumfl ex artery was rapidly removed and placed in ice-cold physiological Krebs solution bubbled with carbogen. The artery was trimmed of connective tissue and cut into 3 mm long segments. While in the dissecting dish, the lower vascular tissue hook (U-shaped hook) was passed through the lumen (Fig. V1). Using forceps, the tissue was then transported to the selected MyoBath and the upper tissue hook was also placed through the lumen of the vessel. Using the micrometer the lower tissue holder was raised until the two prongs of the lower tissue hook were behind the washer of the lower tissue holder. Using a screw driver, the clamp was closed to hold the preparation in place. The MyoBath bracket was used to WORLD PRECISION INSTRUMENTS 11

14 lower the whole preparation into the bath. Vessels were left to equilibrate under zero force for 30 minutes at 37 o C in Krebs solution gassed with 5 % CO2 in O2 and an initial force of 5 grams was then applied using the micrometer. After another 30 minutes, the force was re-adjusted to 5 grams and the tissues were left for a further 30 minutes. 3. Isolation and mounting of spontaneously beating right atria. The following section describes a typical experimental protocol that involves setting up a spontaneously beating rat right atria for study within the MyoBath. Following removal of the rat heart, the right atrium was dissected free and placed in warm, oxygenated physiological Krebs solution. Initial experiments demonstrated that dissection in Krebs solution which was warmed to 37 C and bubbled with carbogen was preferable to dissection in ice-cold Krebs. Under these conditions the atria recovered more quickly and the resting rate was more stable. The isolated right atrium was then impaled with two stainless steel S-shaped atrial hooks (made from 30 1 /2 gauge needles with a sharp tip, see Fig. A1) at each apex. The preparation was then moved to the MyoBath where the top hook was placed over the upper tissue hook. The lower tissue holder was then raised with the micrometer until the lower tissue hook could be placed through the lower tissue hook on the lower tissue holder. The micrometer was then used to apply tension. The whole preparation was then lowered into the 25 ml jacketed organ bath maintained at 37 C. Basal force was maintained at approximately 0.5 gram throughout the experiments. The atrium was washed and left to equilibrate for approximately half an hour until the resting atrial rate was stable. 4. Data acquisition and analysis. The output signals from the Transbridge WORLD PRECISION INSTRUMENTS 12

15 For information on additional tissue holders, see page 14. amplifi er (TBM4) can be recorded either on a chart recorder or through the easy-to-use muti-channel computerized bioscience data acquisition system CVMS. Study of a beating preparation such as the rat right atria requires the use of CVMS. This program is a general purpose data acquisition system that can perform waveform analysis on incoming signals. In the spontaneously beating atria we are interested in the rate (or period), a parameter easily calculated using CVMS. WORLD PRECISION INSTRUMENTS 13

16 Additional Information on Holders and Tissue-mounting There are currently four types of tissue holders available from WPI: atria holder with stimulation feature (AS), vascular holder (V), vascular holder with stimulation feature (VS), and blank holder with stimulation (B). They have been successfully used in the study of cardiac muscle, vascular and non-vascular smooth muscle. AS holder. This holder can be used in the mounting of any small muscle preparation that can be pierced with two S-shaped hooks (made from 30 1 /2 gauge needles), in addition to atria. The S-hooks measure 2 mm in diameter on both ends with a sharp tip on one end where tissue is pieced. The two point stimulation platinum (Pt) electrodes on the holder are 2.54 mm (0.1 in.) apart. B holder. The blank holder is designed to accommodate tissue mounting with hooks of the user s own design. The bottom tissue hook is secured with a nylon screw. Stainless steel wire (0.56 mm or in.) provided in the MyoBath startup kit, can be used to make the top hook which connects to the strain gauge. Platinum electrodes for point stimulation are identical to those on the AS holder. V holder. The vascular holder uses a U-shaped bottom hook and an L-shaped top hook to mount vascular preparations. The hooks are made from 0.48 mm (0.019 in.) stainless steel wire. The U-shaped hook measures 6.35 mm (0.25 in.) in base. For V holders which can accommodate U-shaped hooks of different lengths, call WPI for special order. VS holder. The vascular holder uses the same tissue-mounting mechanism as the V holder, but has two 5 mm fl exible platinum stimulation electrodes which can be positioned around the tissue. The stimulation connector and electrodes are designed to be replaceable. The VS holder can only be used in the 25 ml or 50 ml baths. WORLD PRECISION INSTRUMENTS 14

17 Micro-tissue baths and holders (MH series). WPI now supplies microtissue baths (2 ml or 5 ml) and holders. Instead of tissue-piercing hooks, the MH1 and MHS1 micro-tissue holders use two small clips which clamp onto a tissue strip (clamping force is grams) suitable for mounting tissue strips as small as 0.2 mm in width and 4-10 mm in length. Platinum point stimulation electrodes are available on the holder. Note: If you need special tissue holders or other items for your tissue study, please contact WPI for custom design or modification. Microvessel holders MVS100 (WPI #47120) and MVS200 (WPI #47130) are designed for studies of vessel preparation with sub-millimeter or millimeter diameter, respectively. The spiral platinum electrodes provide effi cient stimulation of a vessel preparation in a current-pipe that is formed between the two spirals. The two electrodes not only can be re-positioned for optimal stimulation but also keep their locations once changed, which can be very useful for repeating experiments. To use the tissue holders, position the two spiral electrodes across the vessel preparation, facing each other in an imaginery current pipe. WORLD PRECISION INSTRUMENTS 15

18 * Electrodes, batteries and other consumable parts are warranted for 30 days only from the date on which the customer receives these items. Warranty WPI (World Precision Instruments, Inc.) warrants to the original pur chas er that this equipment, including its components and parts, shall be free from defects in ma te ri al and workmanship for a period of one year* from the date of receipt. WPI s obligation under this warranty shall be limited to repair or replacement, at WPI s option, of the equip ment or defective components or parts upon receipt thereof f.o.b. WPI, Sarasota, Florida U.S.A. Return of a repaired instrument shall be f.o.b. Sarasota. The above warranty is contingent upon normal usage and does not cover products which have been modifi ed without WPI s approval or which have been subjected to unusual physical or electrical stress or on which the orig i nal identifi cation marks have been removed or altered. The above warranty will not apply if adjustment, repair or parts replacement is required because of accident, neglect, misuse, failure of electric power, air conditioning, humidity control, or causes other than normal and ordinary usage. To the extent that any of its equipment is furnished by a manufacturer other than WPI, the fore go ing warranty shall be applicable only to the extent of the warranty furnished by such other manufacturer. This warranty will not apply to appearance terms, such as knobs, handles, dials or the like. WPI makes no warranty of any kind, express or implied or statutory, including without lim i ta tion any warranties of mer chant abil i ty and/or fi tness for a particular purpose. WPI shall not be liable for any damages, whether direct, indirect, special or con se quen tial arising from a failure of this product to operate in the manner desired by the user. WPI shall not be liable for any damage to data or property that may be caused directly or indirectly by use of this product. Claims and Returns Inspect all shipments upon receipt. Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing. Concealed loss or damage should be reported at once to the carrier and an inspection requested. All claims for shortage or damage must be made within 10 days after receipt of shipment. Claims for lost shipments must be made within 30 days of invoice or other notification of shipment. Please save damaged or pilfered cartons until claim settles. In some instances, photographic documentation may be required. Some items are time sensitive; WPI assumes no extended warranty or any liability for use beyond the date specifi ed on the container. WPI cannot be held responsible for items damaged in shipment en route to us. Please enclose merchandise in its original shipping container to avoid damage from handling. We recommend that you insure merchandise when shipping. The customer is responsible for paying shipping expenses including adequate insurance on all items returned. Do not return any goods to WPI without obtaining prior approval and instructions (RMA#) from our returns department. Goods returned unauthorized or by collect freight may be refused. The RMA# must be clearly displayed on the outside of the box, or the package will not be accepted. Please contact the RMA department for a request form. Goods returned for repair must be reasonably clean and free of hazardous materials. A handling fee is charged for goods returned for exchange or credit. This fee may add up to 25% of the sale price depending on the condition of the item. Goods ordered in error are also subject to the handling fee. Equipment which was built as a special order cannot be returned. Always refer to the RMA# when contacting WPI to obtain a status of your returned item. For any other issues regarding a claim or return, please contact the RMA department Warning: This equipment is not designed or intended for use on humans. World Precision Instruments, Inc. International Trade Center, 175 Sarasota Center Blvd., Sarasota FL Tel: Fax: sales@wpiinc.com UK: Astonbury Farm Business Centre Aston, Stevenage, Hertfordshire SG2 7EG Tel: Fax: wpiuk@wpi-europe.com Germany: Liegnitzer Str. 15, D Berlin Tel: Fax: wpide@wpi-europe.com