United States Patent [19]
`Findley et al.
`
`[II] Patent Number:
`[45] Date of Patent:
`
`4,892,830
`Jan. 9, 1990
`
`[54] ENVIRONMENTALLY CONTROLLED IN
`VITRO INCUBATOR
`
`[75]
`
`Inventors: William Findley; William E. Gibbons,
`both of Houston, Tex.
`
`[73] Assignee: Baylor College of Medicine, Houston,
`Tex.
`
`[21] Appl. No.: 36,061
`
`[22] Filed:
`
`Apr. 2, 1987
`
`Int. Cl.4 ............................................... C12M 1/38
`[51]
`[52] u.s. Cl ......................................... 435/290; 236/3;
`237/14
`[58] Field of Search ...................... 236/3; 237/1 R, 14;
`435/290; 119/37; 312/1
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,955,317 5/1976 Gudin .................................. 435/284
`4,111,753 9/1978 Folsom et al ..•..•.•...•........... 195/126
`4,301,252 11/1981 Baker .................................. 435/290
`4,572,427 2/1986 Selfridge ................................. 236/3
`
`OTHER PUBLICATIONS
`Chetkowski et al., J. In Vitro Fertilization and Embryo
`Transfer, vol. 2, p. 207 (Sep. 1985).
`Auerbach & Brinster, Nature, vol. 217, p. 465 (Feb.
`1968).
`Wright et al., J. Anim. Sci., vol. 42, p. 912 (Nov. 1976).
`
`Brackett & Wililams, Fertil. Steril., vol. 19, p. 144 (Nov.
`1968).
`Gwatkin & Haidri, J. Reprod. Pert., vol. 37, p. 127 (Sep.
`1974).
`Quinn & Harlow, J. Exp. Zool., vol. 206, p. 73 (May
`1978).
`Tervit et al., J. Reprod. Pert., vol. 30, p. 493 (Apr.
`1972).
`Primary Examiner-Henry A. Bennet
`Attorney, Agent, or Firm-Davis Hoxie Faithfull &
`Hapgood
`[57]
`ABSTRACT
`An environmentally controlled incubator is described in
`which the enclosure of the incubator chamber strongly
`attenuates the transmission of light having wavelengths
`below about 500 nm for protecting biological materials
`within the chamber from toxic effects of short wave(cid:173)
`length light. The incubator also includes sensors for
`determining the oxygen and carbon dioxide concentra(cid:173)
`tion within the chamber and means for adding carbon
`dioxide, nitrogen or oxygen to the ambient gas within
`the incubator ion order to maintain the desired levels of
`carbon dioxide and oxygen. A thermostatically con(cid:173)
`trolled heater and a humidification chamber warm and
`humidify the gas as it is circulated through the humidifi(cid:173)
`cation chamber under control of a fan. An airlock is
`provided to allow biological materials to be placed
`within or removed from the chamber.
`
`19 Claims, 3 Drawing Sheets
`
`D
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`
`Hamilton Ex. 1012
`Page 1
`
`

`
`U.S. Patent
`
`Jan. 9, 1990
`
`Sheet 1 of 3
`
`4,892,830
`
`11~
`
`FIG. I
`
`FIG.2
`
`Hamilton Ex. 1012
`Page 2
`
`

`
`·· · U.S. Patent
`
`Jan. 9, 1990
`
`Sheet 2 of3
`
`4,892,830
`
`4/.
`
`17
`
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`
`FIG.3
`
`- 15
`
`FIG.4
`
`FIG. a
`
`Hamilton Ex. 1012
`Page 3
`
`

`
`.
`
`···----
`
`u.s. Patent
`
`Jan. 9, 1990
`
`Sheet 3 of3
`
`4,892,830
`
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`Hamilton Ex. 1012
`Page 4
`
`

`
`1
`
`4,892,830
`
`ENVIRONMENTALLY CONTROLLED IN VITRO
`INCUBATOR
`
`BACKGROUND
`
`30
`
`FIELD OF THE INVENTION
`This invention relates generally to incubators for
`providing a controlled environment for maintaining
`cells, tissues and organisms in vitro during examination
`and manipulation, and more particularly to incubators
`in which the atmosphere and ambient light are con(cid:173)
`trolled to eliminate conditions which may be toxic to
`such cells, tissues and organisms.
`
`2
`formed outside of the culture tubes results in their expo(cid:173)
`sure to air.
`An article entitled Development of One-Cell Ovine
`Embryos In Two Culture Media Under Two Gas Atmo-
`5 spheres by Betterbed et al, at page 547 of Theri(cid:173)
`ogenology, v. 23, n.3(March 1985), reported experi(cid:173)
`ments in which one-cell and two-cell sheep "embryos"
`were cultured in either 5% or 20% 02. The article
`stated that the reduction of oxygen from 20% to 5%
`10 had no effect on embryo development.
`Earlier work reported by W. K. Whitten in a paper
`entitled Nutrient Requirements for the Culture of Preim(cid:173)
`plantation Embryos In Vitro given at a symposium in
`April 1970 in Venice and included in a book entitled
`15 Advances in Biosciences published by Pergamon Press
`found that a higher percentage of mouse embryos
`Advances in vitro fertilization ("IVF") techniques
`~ave !"aised hopes f~r its widespread ~e in permitting
`reached the blastcocyst stage when cultured in an atmo-
`infert~e hum~ patients to beru; children, ~d ~old
`sphere containing 5 to 10% oxygen concentration than
`prormse for stgnificant commerctal benefits m ~al when cultured in higher or lower ambient oxygen con-
`breeding. A problem that has preven~ed the. m?re wtde- 20 centrations.
`Applicants have also observed the toxic effects of
`spread use of IVF, however, ts the ?tsappomtingly low
`pregn:mcy rates t~t have been achievable through the
`oxygen concentration of greater than 10%. Applicants
`techniqu~, even Wtth the transfer of several preem-
`have extended such findings to show that exposures to
`bryos. ":tth humans such ra~es seldom reach 28% and
`20% oxygen, the oxygen concentration of air, for as
`the ong.omg pregnancy ra~e ts ~ually well belo~ 22~. 25 little as two hours or less can also result in toxicity.
`~· Hira~ and R. ~anagunachi have re?~rted ~ thetr
`In addition, applicants have shown that short wave-
`arti~l~ entitled Derr:mental Effec~ of Vzszble Lzght on
`length light as well as atmospheric oxygen concentra-
`Mezoszs of Mamma/zan Eggs In Vztro, J. Ex~ .. Zoo~. v.
`tions is also toxic to mammalian zygotes and pre-
`206, p.365 (1978) that s~ort wavelength vtstb~e hght
`embryos. It is believed that the short wavelength light,
`(below abo~t 480 nm) e~tted from ~uoresce~t ~g~ts of
`such as produced by fluorescent lights, and atmospheric
`the type Wtdely used m laboratones can inhibtt the
`concentrations of oxygen adversely effect both the cui-
`maturational changes (meiosis) which an egg must un-
`ture medium and the zygotes and pre-embryos them-
`dergo before fertilization can occur.
`.
`selves. This phenomenon has not previously been ob-
`Other articles, such as Effect of Room Florescent Lzght
`served in human zygotes and pre-embryos since viable
`on the Deterioration of T~ue Culture Medium by R. L. 35 pre-embryos are normally replaced in the patient after
`two or three divisions, while the deterioration does not
`Wang at page 19 of In Vttro, v. 12 n. 1 (1976), reports
`that tissue culture medium of the type used in cloning
`become apparent until after about 3 or more divisions.
`The above referenced Testart et al article in Fertility
`cell lines deteriorates more rapidly when subjected to
`·
`40 and Sterility provides a partial but unsatisfactory solu-
`fluorescent light.
`The article Apparatus For the In Vitro Fertilization and
`tion to the above noted problems. Storing the culture
`tubes in the dark buckets reduces the exposure to short
`Culture of Human Oocytes by J. Testart et al at pg. 372
`of Fertility and Sterility, v. 38, n.3 (September 1982),
`wavelength florescent light and the controlled atmo-
`acknowledges, as have others, that IVF of mammalian
`sphere within the culture tubes prevents the exposure to
`eggs should be carried out under conditions as close as 45 toxic levels of oxygen while a culture is maintained in
`possible to those occurring in vivo. The article de-
`the tube.
`scribes an incubator in which oocytes and zygotes are
`Using the system, however, a culture is exposed to
`maintained in culture tubes stored in metal buckets so as
`ambient florescent light whenever the culture tube is
`to remain in darkness except when being manipulated or
`removed from the bucket. Additionally, the use of cui-
`examined. A ftltered light source for use with a com- 50 ture tubs strung together by gas supply tubing is a par-
`ticularly inconvenient and disfavored way to maintain
`pound microscope in the incubator is described in
`which the ftlter restricts the illumination to the longer
`cultures which has not found wide acceptance. It is
`wavelength, on the order of 500 to 750 nm, which the
`preferred by most researchers, particularly in the
`article states do not harm mammalian eggs. No ftlter is
`United States, to maintain the zygotes and pre-embryos
`used however with the light source extending into the 55 in culture dishes which are easier to manipulate.
`incubator for a steriozoom microscope. In addition, the
`SUMMARY OF THE INVENTION
`biological materials are exposed to ambient light each
`In accordance with the invention there is provided an
`time they are removed from the buckets.
`incubator for use in maintaining and examining cells,
`The incubator described in the Testart et al article
`also provides a controlled atmosphere for the culture 60 tissues and organisms including mammalian eggs, zy-
`media in the culture tubes by bubbling a controlled gas
`gotes and pre-embryos in culture media which includes
`mixture (5% C02, 5% 02, 90% N2) from a commer-
`an environmentally closed chamber having an enclo-
`cially available premixed gas cyclinder through a bottle
`sure, a portion of which is transparent to light having a
`of distilled water and distributing the humidified gas
`wavelength above about 500 nm and which incorpo-
`through a series of culture tubes. The tubes are stop- 65 rates ftlter means for strongly attenuating transmission
`of shorter wavelength light. Control means are pro-
`pered and the gas is conducted from tube to tube by
`tubing extending through holes in the stoppers. How-
`vided for maintaining the oxygen concentration within
`ever, any examination of the biological materials per-
`the chamber at a level substantially lower than in air.
`
`Hamilton Ex. 1012
`Page 5
`
`

`
`4,892,830
`
`3
`The incubator in accordance with the invention may
`further include a microscope stand and an aperture in
`- the enclosure for permitting a microscope positioned on
`said stand to extend through said aperture, and cuff
`means for sealing between the microscope and said
`aperture.
`In accordance with a further aspect of the invention
`the incubator may include an airlock which may have a
`sliding tray therein for permitting objects, such as cul(cid:173)
`ture dishes to be placed within or removed from the
`incubator during use without contaminating the atmo(cid:173)
`sphere within the incubator with air. According to the
`invention such airlock may include means for coupling
`thereto a source of gas for controlling the composition
`of the gas within the airlock.
`These and other advantages and features of the inven(cid:173)
`tion can be more fully understood from the following
`detailed description of the preferred embodiment of the
`invention illustrated in the accompanying drawings.
`
`25
`
`4
`17 in the front face of the enclosure 15. Plastic cuffs 21
`largely seal the hand openings 17 when not in use and
`seal the annulus between the user's wrist or forearm and
`.the edge of the openings during use to prevent contami-
`5 nation of the atmosphere within the chamber 13 by
`ambient air. Typically, doors 19 (FIG. 1) are provided
`to completely seal the hand openings 17 when they are
`not in use.
`In the illustrated embodiment a binocular microscope
`10 23 is mounted on microscope stage 25 and extends
`through an aperture 27 in the top of the enclosure 15 for
`permitting the user to examine biological materials
`being maintained within the chamber 13. The annulus
`between the microscope 23 and the edge of the aperture
`15 27 is sealed by a flexible plastic cuff 29.
`Other hand openings may be provided in the back or
`side walls of the enclosure 15 as desired. Means such as
`flexible cuffs and doors should be provided to prevent a
`contamination of the atmosphere within the chamber
`20 13. In addition, a door 28 in the rear of the chamber 13
`(FIG. 3) permits the insertion of larger objects, such as
`the microscope 23 into the chamber 13. The door is
`normally kept closed and not used however, when the
`incubator 11 is in use. In some incubators the entire
`enclosure may be lifted off the base for easier access to
`the interior for purposes such as cleaning or installing of
`equipment.
`The gas making up the atmosphere within the cham(cid:173)
`ber 13 is circulated through humidification and temper-
`3D ature control chamber 31located beneath the floor 32 of
`the chamber 13 by means of the fan 33. Gas from the
`chamber 13 is drawn through intake vents 35 in the
`floor 32 and heated by the thermostatically controlled
`heating coil37. The actuation of the heating coil37 may
`35 be controlled by a probe located in the chamber 13, for
`instance, on the microscope stage 25.
`A high humidity is necessary within the chamber 13
`to prevent excessive evaporation of the culture media in
`which the biological materials being maintained in the
`chamber are stored. In order to accomplish this the
`ambient gas, after passing through the heating coil 37,
`passes over a water reservoir 39 for humidification.
`Thereafter the gas passes through the outlet vents 41
`back into chamber 13.
`Most tissue culture media, including those for em(cid:173)
`bryo growth, are formulated to be used in the presence
`of 5% C02, a concentration similar to that found in
`many tissues and biological fluids. In order to maintain
`the elevated 5% C~ concentration in the chamber 15,
`the C02 concentration is sensed by a sensor 43 which
`may, for instance, be an infrared detector of the type
`described in Chapter 10, of the book "Process Analyzer
`Technology" by Kenneth J. Clevett, John Wiley &
`Sons, 1986. When the C02 concentration falls below a
`preset level the sensor 43 causes a solenoid valve 45 to
`open to inject additional C02 into the chamber 13.
`In accordance with the invention it has been found
`that the normal oxygen concentration in air (21%) is
`toxic to mouse zygotes and pre-embryos. Much fewer
`of those zygotes exposed to atmospheric levels of oxy(cid:173)
`gen develop successfully and those that do continue to
`divide often do not progress past the stage normally
`observed at about 72 hours. If the oxygen level is re(cid:173)
`duced to 5-10% however, this inhibition is not ob(cid:173)
`served and the embryos continue to develop. This effect
`has not heretofore been observed in human embyros,
`but it should be noted that such embryos are normally
`replaced within 48 hours which is before the toxic ef-
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is an isometric view of an incubator in accor(cid:173)
`dance with the invention.
`FIG. 2 is a front view of the preferred embodiment of
`an incubator in accordance with the invention.
`FIG. 3 is a top view of the incubator of FIG. 2.
`FIG. 4 is a left side view of the incubator of FIG. 2.
`FIG. 5 is a top view of an interior storage container
`within the incubator of FIG. 2 which includes storage
`space.
`FIG. 6 is a side view of the storage container of FIG.
`
`5.
`
`FIG. 7 is a detail side view of the airlock portion of
`the incubator of FIG. 2.
`FIG. 8 is a top view of the airlock of FIG. 7.
`
`DESCRIPTION OF PREFERRED EMBODIMENT
`Referring now to FIG. 1 of the drawings, there is
`generally illustrated an incubator 11 in accordance with
`the invention. The incubator 11 includes a chamber 13 40
`which is enclosed by a transparent enclosure 15. The
`enclosure 15 is formed of transparent sheet material
`which is formulated to block transmission of short wave
`length light of the type produced by florescent lighting
`commonly used in laboratories. Preferably the material 45
`of the enclosure 15 is chosen to transmit light only with
`a wave length greater than about 500 nm and may be,
`for instance, amber Plexiglass #2422 manufactured by
`Rohm and Haas, Inc., Bristol, Pennsylvania. The amber
`Plexiglass strongly attenuates the shorter wave length 50
`ambient light and so protects culture media and biologi(cid:173)
`cal materials within the chamber 13 from toxic effects of
`short wave length light. The particular wavelength at
`which the material of the enclosure 15 begins to
`strongly attenuate light transmission may be selected in 55
`accordance with sensitivity of the particular biological
`materials and culture media to be stored or examined in
`the incubator 11. Enclosure materials that attenuate
`light of wavelength below about 500 nm are believed to
`be suitable for most applications. For particular applica- 60
`tions it may be possible or necessary to use enclosure
`material having attentation thresholds at wavelength
`below or above 500 nm, respectively.
`FIG. 2 shows a front view of the interior of the incu(cid:173)
`bator in accordance with the invention through the 65
`short wave length light attenuating transparent the
`front panel of enclosure 15. Access by user to the inte(cid:173)
`rior of the chamber 13 is provided by the hand openings
`
`Hamilton Ex. 1012
`Page 6
`
`

`
`4,892,830
`
`5
`fects occuring in mouse embryos could be readily ob(cid:173)
`served. It is believed that the use of air in the incubators
`- of many "successful" IVF programs along with the
`exposure of eggs and embryos to air during their re(cid:173)
`trieval and examination, partially accounts for the rela- 5
`tively low success rates achieved in prior clinical pro(cid:173)
`grams (10-30% pregnancy rate).
`To prevent the effects of oxygen toxicity the incuba(cid:173)
`tor in accordance with the invention injects nitrogen
`(N2) into the chamber 13 whenever the oxygen concen- 10
`tration goes above a preset level in the range of about 5
`to 10% 02. The nitrogen purges out and replaces the
`oxygen until the proper preset level is reached. In the
`illustrated embodiment of the incubator 11 in accor(cid:173)
`dance with the invention, this is accomplished by sens- 15
`ing the oxygen concentration in the gas in chamber 13
`by means of sensor 47 within the chamber 13. The sen(cid:173)
`sor 47 may, for instance, be a commercially available
`zirconium detector of the type described in Chapter 7 of
`the above referenced book "Process Analyzer Techno!- 20
`ogy" or by a commonly used fuel cell detector. The
`zirconium detector is preferred because it is not sensi(cid:173)
`tive to the humidity level within the chamber 13 or to
`the C02 concentration.
`When the oxygen concentration falls below the pre- 25
`selected level in the illustrated embodiment of the in(cid:173)
`vention the sensor 47 opens the solenoid valve 49 to
`inject additional carbon dioxide into the chamber 13
`until the concentration of oxygen is reduced to the
`proper level. The oxygen sensor 47 and the nitrogen 30
`sensor 43 are preferably positioned in the chamber 13
`upstream of the inlet vents 35 in the floor 32.
`In certain applications it may be necessary to gener(cid:173)
`ate oxygen concentrations in the chamber 13 higher
`than in air or to add oxygen to the gas within the cham- 35
`ber to provide a more positive control of oxygen con(cid:173)
`centration. This may easily be accomplished in the incu(cid:173)
`bator in accordance with the invention by providing an
`additional control for adding oxygen under the control
`of the sensor 47 or by replacing the nitrogen source 40
`with an oxygen source.
`Most cultures of biological material are maintained at
`a temperature of about 37" C. and a 90-95% humidity.
`This temperature is the same as the body temperature
`for humans and embryos are, in fact, adversely affected 45
`by exposure to lower "room temperatures". It has been
`reported that a 15 minute exposure of exposure of oo(cid:173)
`cytes (unfertilized eggs) to room temperature for as
`little as 10 minutes results in the disruption of the cellu(cid:173)
`lar apparatus (i.e. spindle fibers) which allows for the 50
`division of the chromosome during cellular division.
`A high humidity is necessary to keep the tissue cul(cid:173)
`ture media from evaporating. This is especially impor(cid:173)
`tant when using C02 and N2 as in the incubator of the
`illustrated embodiment of the invention since these 55
`gases are bottled under extremely dry conditions and
`contain essentially no moisture. Consequently, in the
`absence of a humidification system the atmosphere
`within the chamber 13 would become very dry and
`evaporation of the warm culture media in the culture 60
`dishes would be significant. In the illustrated embodi(cid:173)
`ment of the invention the C02 and N2 are injected into
`the humidification and heating chamber 31 upstream
`from the fan 33. Therefore, before passing into the
`chamber 13 the injected gas is first heated by the heat- 65
`ing coils 37 and humidified by passing over the water in
`the reservoir 39. If desired, apparatus could be provided
`for causing turbulence or atomization of water from the
`
`6
`reservoir 39 in order to increase the humidification
`action. Alternative means of humidification, such as
`bubbling the gas through water may also be used.
`Light for the microscope 23 is provided by light
`source 51 through the fiber optic cable 53. The light
`source 51 is provided with an amber or red filter such
`that the toxic short wave length components are elimi(cid:173)
`nated from the light being supplied to the microscope
`23. The light is delivered to the microscope stage 25
`through fiber optic cable 53 in order to prevent the heat
`generated by light source 51 from adversely affecting
`the temperature regulation within the chamber 13.
`The illustrated embodiment of the incubator 11 in
`accordance with the invention is provided with shelves
`53 and 55 for storing things such as culture dishes
`within the chamber 13. In order to facilitate the circula(cid:173)
`tion of gas within the chamber 13 it is preferable that the
`shelves 53 and 55 be perforated and that they be posi(cid:173)
`tioned with an appreciable gap between their edge and
`the adjacent wall of the enclosure 15.
`It is important when dealing with zygotes and pre(cid:173)
`embryos to maintain constant optimal gas environment
`within the chamber 13. During IVF procedures and the
`examination of embryos it is usually necessary to trans(cid:173)
`fer culture dishes to or from the chamber 15. For in(cid:173)
`stance, during a clinical procedure for the collection of
`eggs, culture dishes or tubes may be transferred as many
`as 20 to 40 times within 45 to 75 minutes. If, as in prior
`art incubators, this is done by means of opening and
`closing a door each time a culture dish is to be inserted
`or removed, undesirable loss of control of gas compo(cid:173)
`nent concentrations, humidity and temperature levels
`are inevitable. It can take substantial amounts of time
`after a door is closed to restore the proper levels and
`C02 and 02. This could have adverse effects on the
`wellbeing of biological materials in the chamber 15.
`Therefore, as seen in FIGS. 2, 3 and 4, there is provided
`an airlock 57 for allowing the easy transfer of biological
`materials in culture dishes and other equipment between
`the chamber 13 and the ambient. As best seen in FIGS.
`7 and 8, the airlock 57 in the illustrated embodiment of
`the invention includes an internal door 59 and an exter(cid:173)
`nal door 61 only one of which can be opened at a time.
`A tray 63 is slidably mounted on tracks 64 within the
`airlock 57. Stops (not shown) may be provided, for
`instance, on the floor of the airlock 57 to limit the exten(cid:173)
`sion of the tray 63 from the open end of the airlock 57.
`The simultaneous opening of both doors 59 and 61 of
`the airlock is prevented in the illustrated embodiment
`by means of microswitches 67 and 69 and electrically
`activated latches 71 and 73. The opening of the interior
`airlock door 59 is sensed by the microswitch 67 which
`in turn activitates the latch 73 mounted on the interior
`top surface of the airlock 57 adjacent the exterior door
`61. The actuation of the latch 73 locks the exterior door
`51 in the closed position. When the interior door 59 is
`closed the microswitch 67 deactivates the latch 73 so
`that the exterior door 61 can be opened. The opening of
`the exterior door 61 is sensed by the microswitch 69,
`which in turn causes the latch 71 to lock the interior
`door 59 closed. When it is desired to insert a culture
`dish 75 into the chamber 13 the exterior door 61 of the
`airlock 57 is opened and the tray 63 is slidably extended
`from the airlock 57 for ease of placing culture dishes
`thereon. After the dishes are placed on the tray 63 the
`tray 63 is slid back within the airlock 57 and the door 61
`is closed. Thereafter the user reaches through the hand
`openings 17 and opens the interior door 59 of airlock 57.
`
`Hamilton Ex. 1012
`Page 7
`
`

`
`7
`The tray 63 is then slid inwardly into the chamber 13
`and the culture dishes 75 may be easily removed for
`-placement on the microscope stage 25 or elsewhere
`within the chamber 13.
`In order to prevent the airlock itself from becoming 5
`the source of the admission of atmospheric air to the
`chamber 13 gas inlet 65 may be provided in the airlock
`57. Preferably commercially available premixed gas
`made up of, for example, 5% 02, 5% C02, and 90% N2
`is infused into the airlock 57 through the connector 65. 10
`Although the airlock 57 is shown in the illustrated
`embodiment as positioned in the back wall of the enclo(cid:173)
`sure 15, it would be equally possible to position the
`airlock elsewhere, for instance, in the left side wall of
`the enclosure 15. The major constraint for the position 15
`of airlock 57 is that a user with hands extended through
`the hand openings 17 should be able easily to operate
`the interior portion of it.
`Although the enclosure 15 in the illustrated embodi(cid:173)
`ment of the incubator 11 in accordance with the inven- 20
`tion has been described as being transparent, if desired,
`portions of it may be opaque and made out of materials
`other than plexiglass. For instance, the portion of the
`enclosure 13 below the floor 32 of the chamber 13 and
`the back wall of the enclosure 15 could be opaque and 25
`made, for instance, out of metal.
`In the embodiment of the incubator in accordance
`with the invention illustrated in FIGS. 5 and 6 of the
`drawings an interior platform and storage compartment
`68 may be provided within the chamber 13 to the right 30
`of the microscope stage 25 for providing additional
`storage space for culture dishes and other items within
`the chamber 13. Preferably the top surface 76 of the
`platform 77 is at the same height as that of the micro(cid:173)
`scope stage 25. The hinged top sections 78 of the plat- 35
`form 77 may be opened for access to the interior storage
`compartment. Apertures in the side walls 79 and 80 of
`the platform 77 permit circulation of gas through the
`interior of the platform for maintaining appropriate
`storage conditions.
`While the invention has been described with relation
`to a particular illustrated embodiment, it should be rec(cid:173)
`ognized that various modifications can be made within
`the scope of the appended claims without departing
`from the spirit and scope of the invention. For instance, 45
`the walls of the incubator chamber could be flexible and
`other equipment could be placed within the chamber 13
`in addition to or instead of a microscope.
`We claim:
`1. An environmentally controlled incubator, compris- 50
`ing:
`(a) an environmentally closed chamber including an
`enclosure having a transparent portion, said trans(cid:173)
`parent portion including means for strongly attenu(cid:173)
`ating transmission of light below a predetermined 55
`wavelength for preventing damage to culture
`media and biological materials within said cham(cid:173)
`ber;
`(b) means for positioning a microscope stage having a
`microscope mounting thereon within said cham- 60
`ber;
`(c) a first aperture for permitting a microscope
`mounted on said stage to extend through said aper(cid:173)
`ture;
`(d) second and third apertures within said enclosure 65
`positioned for allowing one using a microscope
`mounted on said microscope stage to manipulate
`things within said chamber, and sealing means for
`
`40
`
`4,892,830
`
`8
`minimizing leakage between said chamber and the
`external ambient in the annuli between the edges of
`the said apertures and the microscope and forearms
`or hands of a user.
`2. The incubator of claim 1 wherein said transparent
`portion of said enclosures strongly attenuates light hav(cid:173)
`ing a wavelength of less than about 500 nm.
`3. The incubator of claim 1 further including means
`for controlling the concentration of oxygen in the ambi(cid:173)
`ent gas within said chamber.
`4. The incubator of claim 3 wherein said means for
`controlling the concentration of oxygen includes:
`(a) means for sensing the concentration of oxygen in
`said ambient gas; and
`(b) means for adding additional nitrogen to said ambi(cid:173)
`ent gas until the concentration of oxygen in said
`ambient gas reaches a predetermined level.
`5. The incubator of claim 4 wherein said predeter(cid:173)
`mined level is selected to be between about 5 to 10
`percent.
`6. The incubator of claim 1 further including an air(cid:173)
`lock for permitting objects to be placed within or re(cid:173)
`moved from said chamber without modifying substan(cid:173)
`tially the makeup of the ambient gas within said cham(cid:173)
`ber.
`7. The incubator of claim 6 wherein said airlock in(cid:173)
`cludes:
`(a) a cavity extending through said enclosure;
`(b) an internal door between said cavity and said
`chamber;
`(c) an external door between said cavity and the exte(cid:173)
`rior of said chamber; and
`(d) means for permitting only one of said doors to be
`open at a time.
`8. The incubator of claim 7 wherein said airlock fur(cid:173)
`ther includes means for permitting the supply of gas of
`a selected composition to said cavity for maintaining the
`composition of the gas within said cavity to be similar to
`that of said ambient gas within said chamber.
`9. The incubator of claim 4 wherein said airlock fur(cid:173)
`ther includes a tray slidably mounted within said cavity
`for movement between position proximate the internal
`and external sides of said airlock.
`10. An environmentally controlled incubator, com(cid:173)
`prising:
`(a) an environmentally closed chamber including an
`enclosure having a transparent portion;
`(b) means for positioning a microscope stage having a
`microscope mounted thereon within said chamber;
`(c) a frrst aperture for permitting a microscope
`mounted on said stage to extend through said aper(cid:173)
`ture;
`(d) second and third apertures within said enclosure
`positioned for allowing one using a microscope
`mounted on said microscope stage to manipulate
`things within said chamber, and sealing means for
`minimizing leakage between said chamber and the
`external ambient in the annuli between the edges of
`the said apertures and the microscope and forearms
`or hands of a user.
`(e) means for sensing the concentration of oxygen in
`the ambient gas within said chamber; and
`(t) means responsive to the sensing by said sensing
`means of an oxygen concentration above a prede(cid:173)
`termined level for adding additional nitrogen to the
`ambient gas in said chamber for reducing said oxy(cid:173)
`gen level to said predetermined level.
`
`Hamilton Ex. 1012
`Page 8
`
`

`
`9
`11. The incubator of claim 10 wherein said predeter(cid:173)
`mined level is between about 5 to 10%.
`12. The incubator of claim 10 further including an
`airlock for permitting objects to be placed within or
`removed from said chamber without modifying sub- 5
`stantially the makeup of the ambient gas within said
`chamber.
`13. The incubator of claim 12 wherein said airlock
`includes:
`(a) a cavity extending through the enclosure of the 10
`chamber;
`(b) an interior door between said cavity and said
`chamber;
`(c) an external door between said cavity and the exte-
`rior of said chamber; and
`(d) means for permitting only one of said doors to be
`open at a time.
`14. The incubator of claim 10 further comprising:
`(a) a microscope stage;
`(b) an aperture in said enclosure positioned for per- 20
`mitting a microscope mounted on said stage to
`extend through said aperture for use by a user in
`studying biological materials within said chamber;
`(c) means for sealing the annulus between the edge of
`said aperture and said microscope.
`15. The incubator of claim 14 further including a
`storage compartment for storing objects within said
`
`25
`
`10
`chamber and means for pe