A controlled-environment culture system for high resolution light microscopy

Experimental

Cell Research 68 (I 911) 144-148

A CONTROLLED-ENVIRONMENT HIGH RESOLUTION J. A. DVORAK

CULTURE

SYSTEM FOR

LIGHT MICROSCOPY and W. F. STOTLER

Lahoratoqs of Parasitic Discuses, National Institute of Allergy and I~lfwtiorrs Diseccses, and Biomedical Engineering and Instrumentation Branch, Division of Rr.warch Serrimc, National Institutes of Health, Bethesrlrr, Mtl 20014, USA

SUMMARY A culture chamber has been developed within which cells or organisms can be maintained indefinitely in a controlled environment and studied by a variety of light microscopy techniques.

Numerous culture chambers have been devcloped to study living cells or metazoan organisms with the light microscope. The salient features of many continuously perfusable culture chamber designs have been recently reviewed [4]. However, because none of the chambers described to date fulfilled all the requirements of our studies, we designed a system that would have the following characteristics: (1) Usable with all extant transmitted light microscopy techniques including bright field and phase contrast microscopy with high numerical aperture. short working distance objectives and condensers: double beam quantitative interference microscopy; differential interference contrast microscopy; and polarizing microscopy. This necessitates that the assembled chamber have optically flat, strain-free, parallel surfaces with a fixed geometric thickness not exceeding I .2 mm. (2) A completely closed system to safely observe, handle and contain human pathogens. (3) Sterilizability of the assembled chamber Exptl Cdl Res 68

both prior to use and as a means of decontamination at the end of the experiment. (4) Durable construction with biologically inert. non-toxic materials. (5) Design simplicity permitting rapid and easy cleaning and assembly of the chamber. (6) The ability, after removal of the chamber from the microscope, to relocate cells or other objects of interest from a fixed point of reference. (7) Long-term maintenance of optimum physiologic conditions for the cells or organisms being studied. (8) Rapid exchange or replacement of the culture media to observe and study the effects of varying physiologic parameters as well as the instantaneous fixation of the cells for subsequent procedures such as electron microscopy, immunofluorescence, histochemistry or autoradiography. This report describes the design, dimensions and use of a culture chamber which succcssfully fulfills these requirements. Due to the plethora of detail encompassed in actual chamber fabrication, only those dimensions

Controlled-environment

Fig. 1. Component parts of chamber.

which the authors deemed necessary to express their design specifications are reported. However, a complete set of working blueprints will be furnished by the authors on request (cf note 1). Chamber Construction All metal parts unless otherwise noted are fabricated of Type 303 stainless steel. The stainless steel was machined dry without oil lubricants. The culture chamber consists of seven parts (fig. I): A. The holder, with a 19.0 mm lower aperture, a 30.5 mm upper aperture and a total thickness of 8.9 mm. A 1.6mm alignment radius is cut into one edge of the holder (UYYOH’)which fits an alignment pin on the mechanical stage plate of the microscope. Medial to the alignment radius, a serial number is engraved on both faces of the holder. The platform on which the lower

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145

coverglass rests is coated with an 80 pm layer of self-adherent Teflon (note 2). This Teflon layer is required at all steel-glass interfaces to prevent the sharp steel edge from acting as a die and cutting the coverglass under the influence of high perfusion pressures. B., D. Two coverglasses, 25 mm diameter, No. 1i thickness, free of lead oxide. When it is anticipated that polarized light will be used, coverglasses with minimal strain or other optical defects are selected. C. The spacer, with a 19.0 mm aperture and a thickness of 635 pm. Critical control of the thickness of the finished spacer to a tolerance of 25 ,um is essential for proper performance of the assembled chamber. Perfusion ports consist of 27 gauge stainless steel tubing fitted with stainless steel Luertaper hubs.’ The needles pass through and are permanently bonded to the body of the spacer with a solder containing no lead or cadmium (note 3). Both surfaces of the spacer which contact the coverglasses are coated with an 80 pm layer of self-adherent Teflon. E. The pressure plate, with a 19.8 mm aperture and a total thickness of 1.7 mm, has a 2.6 mm wide by 785 pm thick stepped surface which contacts the upper coverglass. This step, which is coated with an180 pm layer of self-adherent Teflon, applies uniform pressure on the coverglass-spacer assembly. F. A silicon c-ring which has its outer diameter ground flat to fit the recessed well of the holder. This o-ring is non-toxic

Fig. 2. Partial cross-sectional assembly drawing of the assembled chamber. 10

711813

Exptl Cell Res 68

146

J. A. Duouak d W. F. Statler

Fig. 3. Assembled chamber inverted on Lucite holder with Teflon 3-way valve, inoculating~syringe and reservoir attached.

barrel and a cotton-plugged glass pressure relief tube. The chamber is inoculated via the Teflon 3-way valve with a tuberculin syringe containing the cell suspension. After inoculation, the chamber is immediately inverted and placed in a Lucite holder (fig. 3). The entire assembly is placed in an incubator until the cells attach to the upper coverglass. After an appropriate time interval, the syringe is removed and replaced with sterile tubing and a 20 ml Luer-Lock syringe containing the desired media. The chamber is flushed with fresh media and placed on the mcchanical stage plate of the microscope for observation (fig. 4). This stage plate is constructed of cadmium-plated steel which allows the use of magnetic clips to hold the chamber firmly in place. Subsequent additions to the chamber (e.g., infectious agents, radiochemicals, etc.) are made through the remaining input port on the Teflon 3-way valve.

and dimensionally stable from -63 to + 232°C (note 4). G. Stainless steel snap-ring (note 5). The spatial relationship of the various parts in the assembled chamber is shown in fig. 2. Culture Procedure

Prior to assembly and use, all metal chamber parts and the silicone :,-ring are ultrasonically cleaned and rinsed. Coverglasses arc cleaned by the HNO,-EDTA technique [3]. After assembly, the input port is fitted with a Teflon 3-way valve (note 6) and the chamber is dry heat or autoclave-sterilized. Following sterilization, disposable sterile tubing (note 7) is connected to the output port of the chamber. The free end of this tubing is connected to a sterile reservoir consisting of a 300 ml Erlenmeyer flask fitted with a silicone stopper containing a 1 ml glass tuberculin syringe Exptl Cell Res 68

Fig. 4. Chamber mounted on mechanical stage plate of microscope. Note the chamber alignment nin on the stage plate, A, the hold-down clips, B,^C, and the perfusion hub stabilizer clips, D, E. These clips have magnetic bases which firmly adhere to the cadmium-plated steel stage plate.

Controlledenvironment A syringe infusion pump (note 8) is used to continuously perfuse the chamber at a constant rate with fresh media. The perfusion characteristics of the chamber are directly related to the flow rate of the media. A flow rate of approx. 1 ml/h results in a clean “sweeping action” of the perfusate through the chamber. Lower flow rates result in diffusion of the perfusate in the chamber: higher flow rates result in vertical mixing. Chamber temperature is maintained with an air curtain incubator (note 9). The relatively large mass of the steel holder and mechanical stage plate act as a thermal buffer, thus restricting temperature fluctuations to approx. 0.2”c. CULTURE

APPLICATIONS

Cells Hhich adhere to a glass surface All cells, both primary and continuous line, used to date have been successfully cultured in the chamber. This includes primary bovine embryo skin, muscle and heart and the derivative lines Vero 161,HeLa [I] and murine chondrosarcoma [2]. Unstimulated mouse peritoneal free macrophages have been maintained in the chamber for 21 days. The culture system has been successfully used to study the interaction of Tr~~punosomu ctxi, the causative agent of Chagas’ disease in man, with unstimulated macrophages and the complete intracellular development of T. c~zr:i in mammalian host cells. Cells. tissues and ougunism H.hich do not udhere to a glass swfmc The cellophane-strip technique for the cultivation of non-adherent cells or tissues [5] can also be used with this chamber. However, for this application the chamber parts must be sterilized prior to assembly and the standard pressure plate replaced with one having a thinner step (635 ,um) to allow for

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147

the added thickness of the dialysis membrane. The high intrinsic anisotropy of the dialysis membrane precludes the use of optical procedures employing polarized light. Two modifications of the cellophane-strip technique as originally described have proved particularly valuable in our work. First, the upper coverglass can be coated with a layer of self-adherent Teflon in which a suitable number of “culture wells” have been cut. The geometry of the wells is dictated by the biological material. Second, the dialysis membrane, which seals the “culture wells” from the main body of the chamber, is hydrated prior to cutting to fit the chamber. This allows the membrane to attain dimensional stability in its final form. Prior to use, the membrane is autoclave-sterilized in distilled water. The introduction of a dialysis membrane into the chamber has no adverse effect upon perfusion. This system has been used to study immune adhesion reactions of parasitic helminths. DESIGN

RATIONALE

Considering the significantly large number of perfusable culture chamber designs for the light microscope which have been reported through the years, an obvious question arises: Why have so many different chambers been necessary? The answer to this question is, unfortunately, complex. Many chambers have been designed for very limited or specialized biological or optical research requirements and are not applicable for other objectives. Other chamber designs have not operationally attained the goals set for them. It is not within the scope of this report to point out the design deficiencies of specific chambers. However, a brief discussion of the design rationale of the chamber described in this report is appropriate. To obtain the desired performance chaExpti

Cell Res 68

148 J. A. Dvorak & W. F. Stotler racteristics previously stated required the careful consideration and interaction of disciplines as diverse as cell biology, mechanical engineering, geometrical optics, metallurgy, chemistry and fabrication technology. The restrictions imposed by the demand that the chamber be usable with all extant transmitted light microscopy techniques dictated not only a fixed geometry of the viewable area of the chamber but its position in the optical path, absence of glass strain and parallel coverglasses. Biohazard requirements could be met only by developing a chamber which could be sterilized fully assembled and utilized reliable permanent seals. Cell biology requirements demanded that all parts of the chamber be certified as biologically inert and non-toxic. Finally, chamber design should not be so complex as to constrain practical use. The chamber reported here successfully fulfills the collective requirements of this approach. The authors express their appreciation to Mr Albert Cam and Mr John Clark for their technical assistance.

NOTES 1. This chamber is commercially available from Nemo Probe, Inc., 7400 Arden Rd., Cabin John, Md 20034, USA. 2. Temp-R-Tape, Type C-400; The Connecticut

Exptl Cell Res 68

3. 4. 5. 6. 7. 8. 9.

Hard Rubber Co., 407 East St., New Haven, Conn. 06509, 06511, USA. All-State No. 430, 90 ‘lo tin and IO o,, silver; The All-State Welding Alloys Co., Inc., P.O. Box 350, White Plains, N.Y. 10602, USA. No. 2-214, Compound S 604-7; The Parker Seal Co.. 17325 Euclid Ave.. Cleveland. Ohio 44112. USA. Truarc No. 5000-I I8 SS420; Waldes Kohinoor, Inc., 47-16 Austel Place, Long Island City, N.Y. 11101, USA. Hamilton Valve No. 3 MFF3; The Hamilton Co., P.O. Box 307, Whittier, Calif. 90608, USA. Bardic No. 1751, Sterile, pyrogen free extension tube; C. R. Bard, Inc., Murray Hill, N.J. 07971, USA. Portable Infusion-Withdrawal Pump, Model 1100; The Harvard Apparatus Co., I50 Dover Rd., Millis, Mass. 02054, USA. Air Curtain Incubator, Model 279; Sage Instruments, Inc., 2 Spring St., White Plains, N.Y. 10601. USA.

REFERENCES I. Gey, G 0, Coffman, W D & Kubicek, M 7, Cancer res 12 (1952) 264. 2. Martinez-Silva, R, Correa, J N, Colon, J 1 & Chiriboga, J, 19th ann meeting of the Tissue Culture Assocn (1968) abst 104. 3. Parker, R C, Methods of tissue culture, 3rd edn, p. 3 I. P Hoeber, New York (1961). 4. Poyton, R 0 & Branton, D, Exptl cell res 60 (1970) 109. 5. Rose, G G, Pomerat, C M, Shindler, T 0 & Trunnell, J B, J biophys biochem cytol 4 (1958) 761. 6. Yasumura, T & Kawakita, Y, Nippon rinsho 21 1963) 1201. Received January 26, 197I Revised version received April 20, I Y7I