U.S. patent application number 09/341025 was filed with the patent office on 2002-01-24 for method and apparatus for culturing cells and tissues.
Invention is credited to VAJTA, GABOR.
Application Number | 20020009803 09/341025 |
Document ID | / |
Family ID | 26068408 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009803 |
Kind Code |
A1 |
VAJTA, GABOR |
January 24, 2002 |
METHOD AND APPARATUS FOR CULTURING CELLS AND TISSUES
Abstract
Cells and tissues, in particular sensitive cells and tissues,
such as oocytes, fertilized oocytes and preimplantation embryos,
which require highly stable physical and chemical environment for
in vitro development, are cultured in closed containers submerged
or immersed in thermostatically controlled liquid baths, the
containers being provided with an appropriate inner atmosphere
containing, e.g. carbondioxide, oxygen and humidity in appropriate
levels. The incubator containers may e.g. be gas and liquid
impervious, flexible, sealable, preferably transparent bags which
after sealing are submerged or immersed directly in the
thermostatically controlled liquid bath. The liquid in the bath is
preferably water. An incubator for submerse or immerse culture of
cells and tissues in the above manner is also described. Also a
transportable liquid incubator for culturing cells and tissues in
the field is described.
Inventors: |
VAJTA, GABOR; (TJELE,
DK) |
Correspondence
Address: |
THOMAS F PETERSON
LADAS & PARRY
224 SOUTH MICHIGAN AVENUE
CHICAGO
IL
60604
|
Family ID: |
26068408 |
Appl. No.: |
09/341025 |
Filed: |
July 15, 1999 |
PCT Filed: |
January 8, 1997 |
PCT NO: |
PCT/DK97/00001 |
Current U.S.
Class: |
435/325 ;
435/289.1 |
Current CPC
Class: |
C12M 21/06 20130101;
C12M 23/14 20130101; C12M 41/22 20130101 |
Class at
Publication: |
435/325 ;
435/289.1 |
International
Class: |
C12N 005/00; C12M
001/00; C12N 005/02; C12M 003/00 |
Claims
1. A method of culturing sensitive cells and/or tissues such as
e.g. oocytes and embryos and other sensitive cells and tissues
derived from multicellular organisms as well as sensitive bacteria
in a container comprising a culture medium, a gaseous atmosphere
and the cells or tissues to be cultured, characterized in that said
container is immersed or submerged so that only the uppermost
top-part of the container is above the upper surface of the liquid
in a thermostatically controlled liquid bath.
2. A method according to claim 1, wherein the sensitive cells or
tissues are oocytes, fertilized oocytes and preimplantation
embryos.
3. A method according to claims 1 or 2, wherein the container is a
vessel in which the culture of the cells and/or tissues is effected
in culture flasks, petri- or well-dishes enclosed in said
vessel.
4. A method according to any of claims 1 or 2, wherein the culture
of the cells and/or tissues is effected in receptacles, e.g. petri-
or well-dishes, enclosed in gas and liquid impermeable, flexible,
sealed bags containing an appropriate gaseous atmosphere and
submerged or immersed directly in the thermostatically controlled
liquid bath.
5. A method according to any of the preceding claims, wherein the
gaseous atmosphere comprises carbondioxide, oxygen and humidity in
appropriate proportions and levels.
6. A method according to any of the preceding claims, wherein
preheated humidified gas or gas mixture is supplied to the
cultivation container.
7. A method according to claims 4, 5 or 6, wherein the cells and/or
tissues, in particular oocytes, fertilized oocytes and
preimplantation embryos, are cultured under limited overpressure of
up to 9 cm water height in at least a part of the culture period by
immersing the bag to the appropriate depth in the liquid bath.
8. A method according to any of the claims 4-7, wherein the bag(s)
in an initial culture period is (are) submerged or immersed into a
liquid bath having a temperature which is lower or higher than that
of another liquid bath to which the bag(s) is (are) transferred
after the initial culture period and the further culture is
effected in a subsequent second culture period.
9. A method according to any of the claims 4-8, wherein the
temperature of the liquid bath in which the bag(s) is (are)
submerged or immersed in an initial culture period is at a first
level and that the temperature of the liquid bath is decreased or
raised to another level in a subsequent second culture period.
10. A method according to any of the claims 4-9, wherein the
composition of the gaseous atmosphere contained in the sealed bag
is changed during the culture period.
11. A method according to any of the claims 4-10, wherein the
gaseous atmosphere in the sealed bag in at least a part of the
whole culture period is expiration air.
12. A method according to any of the claims 4-11, wherein the
thermostatically controlled liquid bath is a transportable thermos
flask provided with heating means and temperature controlling
means.
13. An incubator for culturing cells and/or tissues, comprising a
tank to be filled with a liquid, means for heating said liquid,
means for controlling the temperature of the liquid so as to be
maintained essentially constant at a selected level, and means for
circulating, stirring or agitating the liquid in the tank so that
the temperature of the liquid is essentially the same throughout
the liquid in the tank, characterized in that said tank being
provided with at least one closable roof (cover) having one or more
openings for receiving each a container, basket or rack to be
submerged or immersed in the liquid in the tank, and optionally a
thermal insulating lid for closing each opening in the roof.
14. An incubator according to claim 13, wherein the container to be
submerged or immersed in the liquid bath is a vessel provided with
an insulating cover having an inlet for a gas or gas mixture which
is to constitute the surrounding culture atmosphere in the vessel
and an outlet for releasing excess gas to the surroundings.
14. An incubator according to claim 12, wherein the container is a
gas and liquid impermeable, flexible bag which after sealing is
submerged or immersed directly in the liquid bath of the incubator
tank.
15. An incubator according to claim 14 wherein the bag is
transparent.
16. A combination comprising a transportable liquid bath incubator
for performing culture of cells and tissues in the field and one or
more gas and liquid impermeable, flexible, sealable and preferably
transparent bags, said transportable liquid bath incubator
comprises an inner container filled with a liquid, means for
heating said liquid, means for controlling the temperature of the
liquid so as to be maintained essentially constant at a selected
level, a stopper for closing the upper end of the inner container
and an insulating jacket surrounding the liquid container.
Description
INTRODUCTION
[0001] The present invention relates to a method and an apparatus
for cultivation of cells and tissues. In particular the invention
relates to in vitro cultivation, e.g. maturation, fertilization,
growing, propagation, production and/or maintenance of cells and
tissues, especially sensitive cells and tissues such as e.g.
oocytes and embryos and other sensitive cells and tissues derived
from multicellular organisms as well as certain sensitive bacteria,
yeasts, fungi, molds and mucors which can advantageously be
produced or propagated by the method and the apparatus of the
present invention. Besides, genetically modified cells and tissues
of the above origin, in particular such stemming from multicellular
organisms like mammals and other warm-blooded animals, can be
cultured by the method and apparatus of the present invention.
Furthermore, the use of such cells and tissues for producing
particular desired compounds and materials can also be effected by
the method and apparatus of the present invention.
DESCRIPTION OF THE PRIOR ART
[0002] In vitro culture, e.g. maturation, fertilization, growth,
propagation, production and/or maintenance, of certain sensitive
cells and tissues, in particular such stemming from multicellular
organisms like oocytes and preimplantation embryos, but also
certain bacteria, yeasts, fungi, molds and mucors, and genetically
modified cells and tissues, in particular cells and tissues
stemming from multicellular organisms like mammals and other
warm-blooded animals, require highly stable and constant
environments in order to be successful.
[0003] Thus, the culture medium must contain at least water, salts,
nutrients, essential amino acids, vitamins, hormones and possibly
proteins and growth factors in well defined proportions and levels
as well as a buffer system establishing a strict defined narrow
pH-range and a source of oxygen.
[0004] Usually, the buffer system is a CO.sub.2/bicarbonate buffer
system, the bicarbonate being incorporated into the aqueous culture
medium as from the start of the culturing period but may optionally
be supplemented during the culture period from time to time,
whereas the CO.sub.2 (carbon dioxide) is provided from the
atmosphere surrounding the culture medium during the culture
period. The source of oxygen is usually gaseous free oxygen
(O.sub.2) which is also supplied from the surrounding atmosphere to
the culture medium. Besides, the culture temperature should be kept
within rather narrow limits in order to obtain optimum and/or
successful results.
[0005] Small scale in vitro production, i.e. maturation,
fertilization, growth, propagation etc. as mentioned above, of
sensitive cells and tissues is usually effected in receptacles like
small culture flasks, petri- or well-dishes provided with the
culture medium and the necessary initial cells or tissues. The
receptacles are then placed in an incubator which provide for a
selected constant temperature and an environing atmosphere
containing the gases necessary for development and/or maintenance
of the particular cells or tissues concerned. In particular the
necessary gases comprise humidity (i.e. water vapor), free oxygen
(O.sub.2) and carbon dioxide (CO.sub.2) in specific proportions and
levels.
[0006] Up to now the incubators used for the above production
purposes have been provided in the form of cases or cabinets
provided with insulating thermostate jackets, optionally one or
more shelves for supporting the culture receptacles and dividing
partially the interior volume of the incubator in two or more
compartments, a (usually) vertically hinged front door providing
access to the interior of the incubator, and one or more inlets and
outlets for the gas or gas mixture to be supplied to and released
from (respectively) the interior of the incubator. Such incubators
may have a work space, i.e. an interior volume, of from as small as
about 30-50 liters (except for some special types mentioned below)
to as high as about 350 liters and even more and may be provided
with sensors and control equipments for maintaining the
temperature, humidity, carbon dioxide, oxygen and nitrogen at
preselected levels in the interior of the incubator. The insulating
thermostats jacket may be filled with water in order to provide
easy and even delivery of heat to all points of the walls of the
incubator and to avoid the risk of overheating which can be very
harmful to the cell cultures in the incubator.
[0007] However, each time the front door of the incubator is opened
during a working-day in order to inspect or remove a culture
receptacle already placed in the incubator or to place a new
culture receptacle in the incubator, all the parameters, i.a. the
above mentioned, of the interior of the incubator and of all the
culture receptacles therein are disturbed. The sensors and control
equipments may restore the parameters in the interior volume of the
incubators at the preset levels within about 10 minutes or so, but
in the culture receptacles and media the restoration of the
preselected levels of the parameters concerned may take much longer
time. Furthermore, in as much as the front door of the incubator
may be opened and closed many times during a working-day the
cumulative effect on the development, growth and propagation of the
cultured cells and tissues in the culture receptacles may be rather
significant and serious, i.a. result in decreasing development
and-viability of the cells.
[0008] In particular mammalian oocytes and preimplantation embryos
are considered to be very sensitive to environmental changes. On
the other hand, in vitro production and propagation of such embryos
requires frequent openings of the incubator door for changing or
supplementing the culture media and transferring embryos from one
culture receptacle to another. Professional laboratories working
with in vitro production of embryos start up at least one in vitro
fertilization program per day and try to avoid the above problems
by purchasing a number of expensive CO.sub.2 incubators and/or
regulating the number and duration of openings per day of each
incubator strictly and/or try to keep the temperature of the whole
laboratory containing the incubators at a level as near as possible
to that of the interior of the incubators, i.e. at about
37-39.degree. C. However, the above approach does not solve all the
technical problems concerned and from an economical point of view
it is a very costly approach which is hard to realize for most
laboratories.
[0009] Minimizing the size of the incubators, in particular their
interior working spaces, might be beneficial to embryo development
and has been studied by Boone and Saphiro, 1990; Avery and Greve,
1992; and Gordon, 1994. Analyzing the possible factors affecting
embryo development, Avery and Greve (1992) found no direct
correlation between negative effects on embryo development and
frequency of door openings of large volume incubators and therefore
supposed other factors to be involved, i.e. toxic fumes in the
laboratory air or toxic components originating from the inner
cupper walls of the incubator. However, later observations made by
the present inventor (Vajta, 1994) after a series of unsuccessful
embryo culture experiments in the same type of incubator, but with
inner stainless steel walls instead of copper walls, revealed that
high temperature differences could occur in the partially divided
inner space of large incubators and could persist for hours after
frequent openings of doors.
[0010] Some researchers therefore prefer working with culture
receptacle(s) containing culture medium/media and placed in
commercially available airtight plastic boxes, for example "modular
incubator chambers" manufactured by Flow Laboratories (circular
shape, 30 cm in diameter.times.12 cm in depth), which are placed in
the incubator after filling or refilling them with the desired gas
mixture. However, this requires a more complicated preparation
procedure and the results obtained may vary. The plastic boxes may
eliminate small temperature differences caused by frequent opening
of the incubator door, but the cumulative effects may even be
enhanced.
[0011] Small size double wall stainless steel boxes heated by warm
water circulating in the space between the double wall construction
provide a better solution of the above problems. Such boxes are
commercially available from Henning Knudsen, Hiller.o slashed.d,
Denmark, as K-system boxes. The size, stability and high recovery
rate of gas and temperature levels render these boxes good
candidates for embryo production (Avery and Greve, 1992). However,
the high price of these K-system boxes prohibit most laboratories
from purchasing the necessary optimum quantity of these boxes, i.e.
at least one for maturation and fertilization experiment initiated
and at least one for each continuing experiment. Moreover, even
though the boxes are supplied with pre-mixed gas at a constant flow
rate, recovery rate of the required atmospheric gas composition is
still low and this problem is only partially solved by the possible
manual increase of the flow rate of the gas mixture.
[0012] Single wall K-system boxes are less expensive, but require
precisely heated environment. Placing these boxes in conventional
incubators was up to now the best approach of producing embryos in
an economical manner. However, as heat is transferred to the boxes
by air-conduction, temperature recovery rates are not optimum and
opening of the door of the incubator for working with one culture
will disturb the physical parameters and environments of all the
others in the incubator. Moreover, a single wall box requires a
system to establish the correct gas mixture in each box in the
incubator if optimum conditions shall be maintained within the
boxes, which means high expenses for gas mixture consumption,
control and regulating equipments and provides a source of
complications, errors and troubles.
[0013] WO 85/01514 discloses a method and an apparatus for
harvesting mammalian cells from an artificial substrate, which
method comprises the step of exposing the cells to ultrasonic
energy at a level which is sufficiently high to separate the cells
from the substrate, but which is sufficiently low not to be harmful
to the cells. The ultrasonic energy may be delivered to an external
surface of a culture vessel, e.g. a roller flask dipped in a water
bath having a temperature of approximately 37.degree. C.,
containing the cells to be harvested. However, there is no
disclosure or even a contemplation of culturing or growing the
cells in a vessel immersed or submerged in the water bath before
harvesting the cells.
[0014] U.S. Pat. No. 5,312,630 discloses a method and an apparatus
for preparing an aerobically cultured plant material, such as a
soyfood substrate, inoculated with a beneficial microorganism to
form a cultured food, such as Tempeh. The culture is effected in
trays which are supported on tray racks that are mounted in a water
bath so that the trays are partially immersed in the water bath.
The trays holding the inoculated soyfood substrate are sealed in
the water bath by a cover which mount over the tray racks and is
partially immersed in the water bath to seal the cover over and
around the trays filled with the inoculated soyfood substrate. The
cover can be a single, continuous member covering all the tray
units in the water bath, or there can be individual covers, for
each side-by-side tray unit. An aerating system provides for the
aerobic culturing of the soyfood substrate while a circulating pump
provides a uniform distribution of the water throughout the water
tray. A sensor and a controller actuate a temperature control
system to effect heating and cooling of the water bath as needed to
promote the growth of the microorganisms on the soyfood substrate.
However, this document does not suggest or contemplate the
culturing of sensitive cells and/or tissues like e.g. oocytes and
embryos requiring highly stable and constant environments both as
to pH-range and composition of the culture medium as well as the
composition of the environing atmosphere. Besides, the water bath
is not sufficiently covered or sealed to secure an exact
temperature control of the water in the water bath and thus neither
in the culture trays.
[0015] Therefore, up to now no simple, reliable and inexpensive
method of in vitro production of sensitive cells and tissues has
been available, nor has any simple, reliable and inexpensive
incubator system for such purpose. The present invention remedies
these short-comings of the prior art.
DESCRIPTION OF THE INVENTION
[0016] Thus, the present inventor has discovered that excellent
results can be obtained in the production of sensitive cells and
tissues, specifically oocytes and preimplantation embryos, when the
cells or tissues are cultured in containers, e.g. boxes, vessels or
gasimpermeable bags, submerged or immersed in a thermostatically
controlled liquid bath.
[0017] Hence, the present invention relates to a method of
culturing cells and tissues in a container comprising a culture
medium and a gaseous atmosphere, characterised in that said
container is submerged or immersed in a thermostatically controlled
liquid bath. In particular the cells and tissues cultured are
sensitive cells and tissues.
[0018] In the present description and claims the words "submerged"
or "submersed" are used synonymously and mean that the container
(or any other object specifically mentioned in the context) is or
has been placed at such depth in the liquid bath that a lower part
of the container is below the upper level of the liquid in the
bath, the outer surface of this lower part of the container being
surrounded by and in contact with the liquid in the bath, whereas
the remaining upper part of the container extends above the upper
level of the liquid in the bath. Preferably, only the uppermost
top-part of the container is above the upper surface of the liquid
in liquid bath.
[0019] On the other hand the word "immersed" means in the present
description and claims that the container (or any other object
specifically mentioned in the context) is or has been placed at
such depth of the liquid bath that all parts of the container are
below the upper level of the liquid in the bath, the outer surface
of the container being surrounded by and being in contact with the
liquid in the bath, and no parts of the container extend above the
upper surface of the liquid in the liquid bath.
[0020] The liquid of the liquid bath may be any suitable organic or
inorganic liquid and should preferably be harmless. Examples of
such liquids are mineral, synthetic, vegetable and animal oils,
hydrocarbons, halogenated hydrocarbons, alcohols, esters, ethers
and amides having a sufficiently high boiling point. A few examples
of such liquids are glycerin, ethyleneglycol, ethylacetate,
butylacetate, diethylcellusolve, dioxane, and diethyl- and dimethyl
formamide. Preferably, however, the liquid is water or water mixed
with another liquid or compound miscible with or soluble in
water.
[0021] The liquid may comprise any suitable biocide or antibiotic,
like e.g. a bacteriocide, fungicide, algaecide, acariacide or
insecticide or a mixture thereof, in order to avoid contamination
of the liquid bath with micro organisms or infectant organisms and
thereby minimizing the risk of contamination of the interior of the
culture containers and infection of the cultures contained
therein.
[0022] The liquid bath should be well circulated, stirred or
agitated so that the temperature of the bath is essentially the
same throughout the liquid in the bath at any time. Besides, the
temperature of the liquid should be carefully controlled and
preferably be kept within a range of .+-.1.degree. C. of the
preselected level, more preferably within a range of
.+-.0.5.degree. C., yet more preferably within .+-.0.1.degree. C.,
most preferably within .+-.0.05.degree. C. or less. In fact
commercially available thermostats controlled water baths are
capable of maintaining the temperature of the water in the bath
within a range of .+-.0.05.degree. C. from the preselected level in
the whole bath. This is a very important feature because one of the
most essential factors for in vitro culture of embryos is simply to
control the temperature at any time within the minimum and maximum
of the in vivo occuring physiological levels. Another advantage of
liquid heating is, that overheating, which occassionally occurs in
air-heated systems, may be completely excluded.
[0023] In one embodiment of the method of the invention the culture
of the cells and/or tissues is effected in a vessel submerged in
the liquid bath and provided with a removable cover or lid secured
airtight to said vessel at its top side. Preferably the said cover
is of a heat insulating material and the container is submerged to
such depth in the liquid bath that the top surface of the cover is
above the upper level of the liquid bath and the lower surface of
the cover is below the said level of the liquid bath. Preferably
the cover is provided with at least one inlet for supplying a
preheated gas mixture to the interior of the vessel and at least
one outlet for releasing excess gas to the general surroundings.
The inlet and outlet may be in the form of pipe stubs extending
through the cover to or into the interior of the container and
connected at the outside of the cover with valves and tubes or
hoses. The inlet gas mixture may be preheated by being passed
through a spiral tube immersed in the same liquid bath as the
culture vessel before it enters into the culture vessel. Preferably
such spiral tube is in the form of a thin-walled metal tube having
good thermal conductivity. Also the culture container itself is
preferably made of metal having satisfactory thermal conductivity,
e.g. stainless steel. The cover of the culture container is
preferably made of a non-translucent material when the cells and/or
tissues to be cultured are sensitive to light radiation, but this
will of course prevent direct inspection and observation of the
cultured cells without removing the vessel from the bath or at
least opening the vessel by removing or pivoting the cover of the
vessel.
[0024] A semi-automatic mechanism for filling the vessels with gas
may also be applied. Gas inlets and outlets may be equipped with a
valve: the inlets are open when the gas tube is connected, while
the outlets are open when the pressure inside is higher than
outside. At filling the vessels with a preheated gas mixture, the
tube carrying the gas should be connected to the gas inlet valve,
and the flow of the gas mixture may be started by pressing the
bottom of a time-regulated valve, which stops automatically after 2
to 4 minutes, when the box is completely filled with the gas
mixture.
[0025] In its simplest form the culture medium containing the cells
to be grown is just placed on the bottom of the vessel. However, in
that case only one culture experiment can be effected at a time.
Therefore, in a preferred embodiment the production of cells is
effected in separate receptacles like culture flasks, petri- or
well-dishes optionally enclosed in sealed bags of plastic film,
metal foils or more preferably laminates of such materials. These
receptacles may be placed on the bottom of the vessel or more
expedient on the shelves of a rack which is placed in the vessel.
In the latter case a water layer can be provided on the bottom of
the container for maintaining sufficient humidity in the interior
of the culture container.
[0026] It has been discovered that excellent results are obtained
when culturing cells with the above embodiments of the method of
the invention, apparently because it is possible to maintain the
culture conditions, in particular the temperature and the
composition of the surrounding atmosphere, stable and constant to
an extent not obtainable hitherto by any other method or equipment.
Besides, manipulation of one vessel does not at all affect the
physical parameters and compositions of the environments of the
other culture vessels in the same liquid bath. Finally, infections
occasionally occurring in tissue cultures, especially at primary
cultures as embryo culturing, cause minimal problems with LBI:
removable boxes may be cleaned, sterilized without disturbing the
function of the whole device.
[0027] In a further embodiment of the method of the invention the
culture of cells and/or tissues is effected in a receptacle, e.g. a
petri- or well-dish, containing a culture medium and enclosed in an
airtight sealed flexible bag provided with an appropriate
atmosphere, which bag is submerged or immersed directly in a
thermostate controlled liquid bath. In this embodiment the bag is
expediently placed in a foramenious, grid or wire mesh basket or a
rack with grid or wire mesh shelves in order to lower the bag to
the desired depth of the liquid bath. The baskets and racks may be
made of metal or plastic.
[0028] The appropriate gas mixture is passed into the bag before
sealing it or it can be passed into the bag after its sealing when
the bag is provided with an inlet for supplying the desired gas or
gas mixture and an outlet for releasing excess gas to the
surroundings. The in- and outlet should be provided with valves or
other appropriate means, e.g. clamp or stop cocks or hot sealing
facilities, for closing the access to the bag hermetically.
Alternatively, a presealed bag can be filled with the proper gas
mixture through a sterile injection needle passed through the wall
of the bag into its interior volume whereupon the needle is
retracted and the puncture hole in the bag is sealed. The culture
receptacle containing the cell(s) to be grown is in the former and
latter cases always and in the middle case preferably introduced
into the bag before sealing it.
[0029] The bag to be used is optionally manufactured of a
transparent plastic film or laminate allowing inspection and
observation of the cultured cells during the culture period without
breaking the sealed bag and without or with only minimum
disturbance of the cultured cells and their environmental
conditions. However, the bags to be used may also be manufactured
of a non-translucent film material, e.g. a plastic film laminate
comprising a metal foil, e.g. of aluminium. Such bag are particular
suitable for culturing light sensitive cells and tissues for
prolonged periods of time.
[0030] If the culture medium and cells to be grown are placed in a
receptacle like a well- or petri-dish which in turn is introduced
into a transparent plastic film or laminate bag which is then
sealed, a thin layer of a sterile liquid, preferably oil, could be
used to keep the inner surface of the bag attached to the surface
of the culture dish to increase optical clarity for direct
microscopic examination. In this way direct observation and
examination is possible at most magnifications. According to
experiments performed by the inventor, a limited number (at least
up to 5) of investigations and photographings does not decrease the
development of embryos (see Pilot experiment 6). When a bag is
placed on a preheated microscopic stage and covered with a
transparent plastic or glas box to increase heat-stability,
microscopic observation in this manner can be continued for
extended periods of more than one week.
[0031] Besides, culturing sensitive cells and tissues, in
particular mammalian oocytes and preimplantation embryos, in a
culture medium introduced into a flexible bag provided with an
appropriate environing gas mixture, which is then submerged or
immersed in a thermostate controlled liquid bath, has proved to
provide some quite unexpected advantages and possibilities.
[0032] Thus, if the culture of cells and/or tissues is effected in
air-filled bags (i.e. containing the appropriate gas mixture) the
system is uniquely suitable to perform experiments under limited
pressure exactly defined by the depth of immersion of the bags into
the liquid of the bath. Limited overpressure might have beneficial
effects on development of certain tissues as well as embryos of
certain species (see Pilot experiment 2a). On the other hand it has
been proven that overpressure occurring at up to 9 centimeter water
depth (from the surface) has no harmful effect on the development
of bovine embryos (see Pilot experiment 2b).
[0033] Another particular advantage of this system is that the bags
can be submerged or immersed for a limited period into a liquid of
a temperature lower or higher than the physiological one, for
example to activate enucleated oocytes at +10.degree. C. for
cloning by nuclear transfer. A rapid and well controlled change of
the temperature can then be obtained in a very simple way without
disturbing the appropriate gas atmosphere surrounding the cultures,
e.g. by transferring the bag to another liquid bath incubator
having a different temperature or by suddenly changing the
temperature of the liquid in the same liquid bath incubator. (For
need of such temperature changes see P. Chesne et al., 1993).
[0034] Furthermore, as the flexible bags can be filled with an
unlimited variety of gas mixtures, the system provides a very
simple possibility for performing cell, tissue or embryo cultures
in specifically composed gas mixture atmospheres or for making
comparative experiments investigating the effect of different
levels of certain components of the culture atmosphere. For bovine
embryo maturation and fertilization, for example, a gas mixture
consisting of 5% carbon dioxide in air is used by many laboratories
worldwide. However, for embryo culture certain culture forms
require a gas mixture consisting of 5% oxygen, 5% carbon dioxide
and 90% nitrogen. Other cultures have to be performed in 5% carbon
dioxide in air, 2% carbon dioxide in air, etc. (For need of such
different culture atmospheres see Y. Fukui et al., 1991).--So far,
no simple and inexpensive equipment offering the flexibility of the
present invention in this field has been available.
[0035] Experiments performed completely in the sub- or immersed bag
system have led to the discovery that the optimum composition of
the surrounding atmosphere used in the system for culturing bovine
embryos is lower in carbon dioxide content than the 5% carbon
dioxide in air used previously. The percentage of oocytes
developing to the blastocysts stage is significantly higher when
the carbon dioxide level is lowered to 3,5% (see Pilot experiment
3).
[0036] The cultivation of the sensitive cells and tissues like
oocytes and embryos in air-filled flexible bags has the further
advantage that the initiation of the culture, i.e. initial growth
of the cells, need not to be started in a laboratory having the
necessary complicated and sophisticated laboratory equipments, but
can be commenced on location in the field.
[0037] Thus, in addition to flexible sealable bags the only further
equipment necessary for initiation of the maturation of oocytes
removed from an animal on a farm in the country is just a
relatively simple (preferably electrically) heated and temperature
controlled thermos flask or container filled with a liquid,
preferably water, of the appropriate temperature. Such thermos
flask, bottle or container can be electrically powered from a
battery, an accumulator or a car cigaret lighter plug. The
necessary gas mixture to be loaded into a bag before sealing it
after introduction of culture medium, oocyte(s) can simply be
expiration air blown into the bag by a person, e.g. the researcher.
Expiration air has a rather constant CO.sub.2 level of about 4% (by
volume) and an O.sub.2 level of about 16% (by volume) and it has
been established that cultivation of fertilized bovine oocytes can
be performed in a period of at least seven days without need for
breaking the sealed bag and replenish, modify or exchange neither
the culture medium nor the environing atmosphere provided in this
way, i.e. until the development of the blastocyst stage has
occured. Comparing development of fertilized oocytes performed
either in 4% carbon dioxide in air stemming from a compressed air
bottle or expiration air, in a laboratory water bath incubator with
high temperature stability disclosed no significant differences
(see Pilot experiment 4). Experiments performed for the whole
culture period (7 days) in a transportable thermos flask with
slightly less accurate temperature stability resulted also in good
blastocyst rates (see Pilot experiment 5).
[0038] The above embodiment of the invention allows not only
embryology work to be initiated on location in the field, but also
performance of culture of sensible cells and tissues nearly
anywhere, e.g. in connection with sampling in the jungle,
wilderness, desert or the arctic or antarctic areas of the
Globe.
[0039] The present invention also comprises equipment for
performing the method of the invention.
[0040] Thus, the invention also relates to an incubator for
culturing cells and tissues, comprising a tank to be filled with a
liquid, means for heating said liquid, means for controlling the
temperature of the liquid so as to be maintained essentially
constant at a selected level and means for circulating, stirring or
agitating the liquid in the tank so that the temperature of the
liquid is essentially the same throughout the liquid in the tank,
said tank being provided with a roof (i.e. cover) having one or
more openings for receiving each a container, basket or rack to be
submerged or immersed in the liquid in the tank, and optionally a
thermal insulating cover to close the opening after sub- or
immersion of the container(s), basket(s) or rack(s) in the liquid.
The tank should preferably be thermal insulated by an insulating
jacket and the roof should preferably be made of a thermal
insulating material.
[0041] The container to be sub- or immersed in the tank liquid can
be a vessel provided with a lid secured airtight thereto and
wherein at least one inlet is passed through the lid for supplyig a
preheated gas mixture to the interior of the vessel and at least
one outlet is passed through the lid for releasing excess gas to
the surroundings. The inlet and outlet may be in the form of pipe
stubs extending through the lid to or into the interior of the
container and connected at the outside of the lid with valves and
tubes or hoses. The inlet tube is connected to a heating means,
preferably a tube immersed in the same liquid bath as the culture
vessel so as to heat the entering gas mixture to essentially the
same temperature as that of the liquid bath before it is passed
into the culture vessel. Preferably such tube is in the form of a
spiral shaped, thin-walled metal tube having good thermal
conductivity. Also the culture vessel itself is preferably made of
metal having satisfactory thermal conductivity, e.g. stainless
steel. The lid of the culture vessel can be made of a transparent
material so that inspection and observation of the cultured cells
can be effected without removing the vessel from the bath and even
without opening the vessel by removing or pivoting the lid of the
vessel, but when the cells or tissues to be cultured are sensitive
to light radiation the lid should be made of a non-translucent
material or it should be covered with a layer or sheet of such
material.
[0042] The vessel may have any suitable shape, e.g. box shaped,
spherical or cylindrical shaped or supereliptical shaped, but is
preferably box-shaped with rounded comers so as to facilitate
cleaning of the vessel.
[0043] The lid of the vessel should be manufactured of a thermal
insulating material and either be double-walled or be of a
sufficient thickness. Preferably it is made of a plastic
material.
[0044] Baskets or racks provided with shelves may be placed in the
vessels for supporting separate culture receptacles like culture
flasks, petri- or well-dishes or sealed bags containing the culture
medium and the cells to be grown. Such baskets and racks can be
manufactured of a plastic material or a metal, in particular
stainless steel. The baskets and shelves are preferably made of
wire mesh or gratings of the above materials.
[0045] The container to be sub- or immersed in the thermostate
controlled liquid in the tank can also be in the form of an
air-tight sealable flexible bag which is to enclose a receptacle
filled with culture medium, the cells or the tissues to be grown
and an appropriate gas or gas mixture and then sealed.
[0046] The inside of the bag may be provided with a film or a layer
of a heat sealable material so that the bag can simply be
hermetically closed by heat sealing a stripe across the bag near
its open end. In case the bag is to be formed of a flexible film
tube provided with an inner heat sealable film material, the tube
will have to be cut into appropriate lengths, the ends of which
will have to be heat sealed, usually first a bottom end and then a
top end. The bag can also be manufactured of a sheet of such film
material by cutting it into appropriate rectangular pieces which
are then folded along a central line, heat sealed along the two
side edges and finally at its top edge (after introduction of the
culture receptacle). Alternatively, the bag can be manufactured of
a laminate film sheet material having a heat sealable film layer
adhered to one of its surfaces by cutting appropriate rectangular
pieces of such material and placing two such rectangular pieces on
top of each other, the heat sealable layers facing each other, and
then effecting heat sealing along three of the edges of the whole
assembly, the fourth remaining unsealed until use of the bag.
[0047] The flexible sealable bag may, however, also be provided
with a pressure sensitive adhesive attached circumferentially along
a stripe on the inside surface of the bag at or near its open
end(s), which adhesive stripe is initially covered with a slip tape
or strip which upon removal makes it possible to seal the bag
air-tight by effecting a calendering pressure on the outside of the
bag along the adhesive stripe area inside the bag.
[0048] The bag may be provided with an inlet for supplying the
desired gas or gas mixture to the bag and an outlet for releasing
excess gas to the surroundings. The in- and outlet may be in the
form of pipe stubs provided with or connected to valves or other
appropriate means, e.g. clamp or stop cocks or hot sealing
facilities, for closing the accesses to the bag hermetically.
[0049] The bag should be made of a gas impermeable material and
should also be impermeable to and insoluble in the liquid in the
tank. Optionally the bag is manufactured of a transparent plastic
film or laminate material allowing inspection and observation of
the cultured cells enclosed in the bag. Films sold by Rexam
Metallising under the trade name Camclear Polyester Laminate 12/45,
Anti-Fog, have proved to be particular suitable. Dew gathering on
the inside of bags made of this film material, which may prevent a
clear view of cells or tissues enclosed in the bags, can be
prevented by oiling the inner surface of the bags or by treating it
with other suitable anti-fog or liquid means. However, a metal foil
can be included in the laminate material in order to complete its
impermeability to gas and liquid, but this will of course prevent
direct visual observation of the cells enclosed in the bags.
[0050] The culture medium and cells to be grown may be introduced
directly in the bag which is then sealed after introduction of the
appropriate gas mixture. However, the culture medium and cells to
be grown are preferably placed in receptacles like well- or
petri-dishes which is then introduced into the bag, which is
supplied with the appropriate gas mixture and then sealed.
[0051] Even though it is advantageous that the bag and the optional
receptacles contained therein are transparent so as to make it
possible to inspect and observe the cells and culture medium during
the culture period it may be advantageous too to cover the opening
in the roof of the tank after sub- or immersing the bag in the
liquid in the tank with a non-translucent cover, because the cells,
in particular oocytes and preimplantation embryos, are sensitive to
light and other form of radiation.
[0052] The invention also comprises a transportable combination kit
for performing cell and tissue culture on location in the field,
said combination kit comprises a (electrically) heated and
thermostate controlled thermos container, flask or bottle filled
with an appropriate liquid, in particular water, and air-tight,
flexible, sealable bags and optional culture receptacles like
petri- and well-dishes. The thermos container, flask or bottle can
be electrical powered from a battery, an accumulator or a car
cigaret lighter plug. If optimum gas mixtures are not available
under field conditions expiration air from the lungs of a person
can be used instead.
[0053] The invention will now be described in further details with
reference to the examples given below and the accompanying
drawings, wherein:
[0054] FIG. 1 is a top view of an embodiment of a liquid bath
incubator according to the present invention covered with a roof
containing openings for submerging both vessels and basket(s)
and/or rack(s) in the liquid in the incubator tank;
[0055] FIG. 2 is a sectional side view of a box-shaped vessel in
the tank of the incubator shown in FIG. 1;
[0056] FIG. 3 is a sectional side view of a box-shaped vessel in
the tank of the incubator shown in FIG. 1;
[0057] FIG. 4 is a sectional end view of the vessel shown in FIG.
3;
[0058] FIG. 5 is a sectional side view in part showing the
gas-supplying system for the culture vessels of the incubator shown
in FIG. 1;
[0059] FIG. 6 is a perspective view of an embodiment of a flexible,
sealable bag for performing sub- or immersed culture of cells and
tissues according to the present invention;
[0060] FIG. 7 is a perspective view of another embodiment of a
flexible, sealable bag for performing sub- or immersed culture of
cells and tissues according to the present invention, said bag
being provided with in- and outlet for supplying gas mixture to and
releasing excess gas from the bag;
[0061] FIG. 8 is a sectional view of a transportable liquid bath
incubator for performing culture of cells and tissues in the
field;
[0062] FIG. 9 is a bar chart showing the comparative results of
embryo development in a liquid bath incubator (LBI) according to
the present invention and in a Salvis.RTM. incubator (details
explained in Pilot experiment 1);
[0063] FIG. 10 is a bar chart showing the results of embryo
development in air-tight flexible laminate foil bags filled with
expiration air and immersed to either 1 cm or 5 cm below the upper
water level in an LBI of the present invention (details explained
in Pilot experiment 2a),
[0064] FIG. 11 is a diagram chart showing the comparative results
of embryo development as explained above under FIG. 10, but being
immersed to depths of 0, 5, 9 or 13 cm below the upper level of the
water in an LBI of the present invention (details explained in
Pilot experiment 2b);
[0065] FIG. 12 is a bar chart showing the comparative results of
embryo development in air-tight flexible laminate foil bags filled
with a mixture of either 3.5% or 5% carbon dioxide in air and
immersed to the same depth of the water in an LBI of the present
invention (details explained in Pilot experiment 3);
[0066] FIG. 13 is a bar chart showing the comparative results of
embryo development in air-tight flexible laminate foil bags filled
with either expiration air or 4% carbon dioxide in air and immersed
to the same depth of the water in an LBI of the present invention
(details explained in Pilot experiment 4a); and
[0067] FIG. 14 is a bar chart showing the results of 25 embryo
development experiments performed in air-tight flexible laminate
foil bags filled with expiration air and immersed to the same depth
of the water in an LBI of the present invention (details explained
in Pilot experiment 4b).
DETAILED DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 shows a top view of an embodiment of an incubator 10
according to the present invention. 20 indicates vertical side- and
endwalls of a box-shaped tank having an open top side, an
insulating jacket (not shown) surrounding the tank on its end, side
and bottom walls may be provided. The tank is covered by a roof 40
having rectangular openings 50 for four box-shaped vessels to be
submerged in a liquid contained in the tank. The vessels have a
flange extending horisontally from the upper edge of the vessels to
engage with the edges surrounding the openings in the roof so as to
support the vessels thereon. 55 indicates an opening in the roof
for submerging or immersing racks or baskets containing sealed bags
directly in the liquid. Particular means may be provided for
lowering these racks and baskets to specific depths from the upper
level of the liquid in the incubator tank. 60 Indicates a combined
temperature control, heating and circulator means to agitate the
liquid in the tank, e.g. a stirrer or a propeller, so as to
maintain an essentially constant temperature throughout the bath.
70 indicates a gas regulating unit. The liquid in the tank will
preferably be water. When all the vessels are inserted in the
openings in the roof of the tank there will be no direct access to
the liquid in the tank because all openings in the roof will be
covered. Openings 50,55 in the roof not occupied by inserted
vessels will be covered with particular lids (not shown).
[0069] FIG. 2 is a sectional side view of a box-shaped incubator
vessel. 80 indicates the vertical end walls of the vessel and 81
indicates a cover or lid at the top of this vessel. 82 indicates an
inlet, e.g. a pipe stub to be connected with a tube or hose and
optionally a valve (not shown), for a preheated gas or gas mixture.
83 indicates an outlet, e.g. a pipe stub provided with a valve and
optionally connected to a tube or hose, for releasing excess gas
from the interior volume of the vessel to the surroundings. 84
indicates an air-tight insulation strip arranged between a flange
on the vessel lid 81 and a flange at the top of the vertical vessel
walls 80. 85 Indicates a handle for lifting or lowering the lid or
the whole vessel. Clamps or screws (not shown) may be provided
along the flanges of the lid and the vessel for maintaining a firm
air-tight sealing therebetween. 95 indicates the upper level of a
water layer placed on the bottom of the vessel for providing
maximum humidity inside the vessel.
[0070] FIG. 3 is a sectional side view of another box-shaped vessel
in the tank of the incubator shown in FIG. 1. 80 indicates the
vertical end wall of the vessel and 81 indicates the cover at the
top of this vessel. 82 indicates an inlet, e.g. a pipe stub to be
connected with a tube or hose and optionally a valve (not shown),
for a preheated gas mixture. 90 indicates a rack with shelves 91
placed in the vessel for supporting culture containers, e.g.
culture flasks, petri- or well-dishes or flexible, sealed bags. 95
indicates the upper level of a water layer placed on the bottom of
the vessel for providing maximum humidity inside the vessel. 100
indicates the upper level of the liquid in the incubator tank.
[0071] FIG. 4 is a sectional end view of the vessel shown in FIG.
3. 80,81,82,90,91,95 and 100 have the same meaning as in FIG. 3 and
83 indicates an outlet, e.g. a pipe stub pro- vided with a valve
and optionally connected with a tube or hose, for releasing excess
gas from the interior of the vessel to the surroundings.
[0072] FIG. 5 is a sectional side view in part of the incubator
shown in FIG. 1, which shows the gas-supplying system for the
culture vessels submersted in the incubator tank. 10 indicates the
entire incubator, 20 the incubator tank, 30 the insulating jacket
surrounding the tank, 70 the gas regulating unit comprising a time
regulated valve (TRV) and a pressure regulator (PR). 71 indicates a
tube supplying gas or a gas mixture from a source therefore (not
shown), 72 indicates a tube coil (preferably of a thin-walled metal
tube) for preheating the gas or gas mixture to be supplied to the
culture vessels 80. 73 indicates the outlet from the tube coil
passing preheated gas or gas mixture from the coil to the inlet(s)
82 of the incubator vessels 80. As previous 81 indicates a thermal
insulating lid at the top of the incubator vessel.
[0073] FIG. 6 is a perspective view of an embodiment of a flexible,
sealable bag for performing sub- or immersed culturing of cells and
tissues according to the present invention. 110 indicates the bag,
112 indicates a heat-sealed stripe across the bottom end of the
bag, 115 indicates the opening of the bag at its top end before
sealing it, and 111 indicates a heat sealable film binded to the
inside of the bag. When the culture medium and the cells to be
cultured have been introduced into the bag, either directly or in a
receptacle like a petri- og well-dish, an appropriate air mixture
will repeatedly (4-5 times) be blown into the bag and expelled by a
slight mechanical pressure applied on the outside of the bag to
ensure complete exchange of the gas surrounding the culture and the
bag will then be hot sealed along a stripe across the bag near its
top end 115. The bag will then be ready for sub- or immersing it
into the liquid of a liquid incubator.
[0074] FIG. 7 is a perspective view of another embodiment of a
flexible bag for performing sub- or immersed culturing of cells and
tissues according to the present invention. 110, 112 and 115 have
the same meaning as explained for FIG. 9. 114 indicates a pressure
sensitive adhesive stripe at the inner surface of the bag, which is
covered by a slip tape or strip. When sealing the open top end of
the bag the slip tape or strip is simply pulled off and the bag is
sealed by subjecting the outside surface of the bag at its open end
to a firm calendering pressure. 116 indicates an inlet, e.g. in the
form of a pipe stub, for introducing a culture gas or gas mixture
into the bag after its sealing at its open top end 115. 117
Indicates a ring-shaped sealing stripe fastening the inlet tube to
the bag. 118 indicates an outlet for excess gas in the bag, which
gas will be released to the surroundings. Also the outlet may be in
the form of a pipe stub, and 119 indicates a ring-shaped sealing
stripe fastening the outlet 118 to the surface of the bag. When
culture medium and cells have been introduced into the bag,
optionally in a receptacle like a petri- or well-dish, the bag will
be rinsed by blowing the culture gas or gas mixture through the
inlet 116 and out through the outlet 118. When the interior of the
bag has been thoroughly rinsed in this manner the inlet and outlet
will be closed, e.g. by valves (not shown) or clamps (not shown) or
by heat-sealing the pipe stubs 116 and 118. The sealed bag is then
submerged or immersed in the liquid of a liquid incubator of the
present invention.
[0075] Instead of the inlet 116 and outlet 118 the bag can be
provided with one or more self-sealing membranes 120, e.g. rubber
septums, sealed to the wall of the bag with a ring-shaped sealing
stripe 121 or optionally with a circular sealing layer provided
between the membrane and the wall of the bag. The presealed bag can
then be filled with the proper gas mixture through a sterile
injection needle passed through the membrane into the interior
volume of the bag. When retracting the needle the puncture hole
made by the needle will be closed automatically due to the
selfsealing membrane material.
[0076] FIG. 8 is a sectional view of a transportable liquid bath
incubator for performing culture of cells and tissues in the field
according to the present invention. In principle this incubator
consists of a thermos container 130 comprising an inner container
131, an outer container 132 and a heating jacket 133 therebetween,
expediently containing a heating medium, e.g. water or another
suitable liquid. Electrical heating means 135, e.g. an isolated
metal coil or helix is provided in the jacket for heating the
jacket medium to a selected temperature controlled by a thermostats
sensor (not shown). The electric heating means is supplied with
electric power by the electric cable 134 which in turn is connected
to an electrical power supply, e.g. a battery, an accumulator or a
car cigaret lighter plug (not shown). 136 indicates the incubator
liquid, preferably water, and 110 indicates a sealed culture bag
immersed in the incubator liquid. 138 indicates a cover plug
provided with a thermal insulating lower part 139. 140 indicates a
handle for pulling up the cover plug. The cover plug 138,139 may
also be provided with threads to be engaged with corresponding
threads at the inside (or outside) of the container 130 at its top.
In addition to the heating jacket 133 a further insulating jacket
comprising an insulating material like glass-wool can be arranged
around and enclosing the outer container 132.
[0077] In some respects the transportable liquid incubator may
resemble a thermos bottle or flask except that it must be provided
with heating means and a thermostate controlling means providing
for strict control of the temperature of the incubator liquid.
[0078] Pilot Experiment 1.
[0079] Embryo Production
[0080] Maturation of bovine oocytes, fertilization and culture of
embryos has been described in detail elsewhere (Vajta et al, 1995).
Briefly, oocytes were aspirated from slaughter-house-derived
ovaries, matured for 24 h in TCM-199 medium supplemented with 15%
calf serum, eCG and hCG. Insemination (Day 0) was performed with
frozen-thawed, Percoll-selected sperm. After 22 h, zygotes were
vortexed, then cultured on the granulosa cell monolayer formed
spontaneously in the maturation dishes in TCM-199 supplemented with
5% (15% from Day 5) calf serum. Cultures were evaluated on Day 7
and 8 after insemination. For maturation and fertilization, which
processes require relatively short incubation time, a double-wall
K-system box with continuous gas flow and heated by an external
circulator (Henning Knudsen Engineering, Hiller.o slashed.d,
Denmark) was used. Embryo cultures were performed in the
thermostates described below.
[0081] Liquid Bath Incubator (LBI) Used for Present Experiments
[0082] A simple preliminary model of an LBI was constructed and
used for the preliminary experiments. A large covered plastic
container (W.times.D.times.H: 380.times.310.times.180 mm) filled
with water was heated by a HETO DT circulator placed in the center
of the cover. In the comers, four cylindrical shaped plastic boxes
(DxH: 115.times.135 mm) with airtight and thermoinsulated caps were
inserted. However, a 30 mm part of the plastic boxes, partially
uninsulated, was above the water level. Two plastic tubes with 1 mm
inner diameter were fixed through the caps for gas delivery.
Opening and closing of these tubes as well as starting and stopping
gas flow was performed manually. Approx. 1 m of the plastic gas
tube was immersed into the water to achieve preheating of the gas
mixture.
[0083] Thermostate Used for Control Experiments
[0084] Four single-wall metal boxes (W.times.D.times.H:
160.times.150.times.80 mm); (K-system, Henning Knudsen Engineering,
Hiller.o slashed.d, Denmark) were placed in a Salvis Thermocenter
(Salvis, Luzern, Switzerland) in two vertical lines. Vertical
airtight doors were placed on the front wall, and pre-heated
humidified gas was delivered continuously to ensure the appropriate
environment.
[0085] Comparison of Parameters
[0086] Approximate volume of suggested metal boxes and used plastic
boxes was the same, about 1400 cm.sup.3. Filling these boxes with
gas mixture required no more than 2 min. at appr. 0.1 bar
pressure.
[0087] Temperature of the gas reaching the boxes was 31-32.degree.
C., which was unsatisfactory. However, using a spiral metal tube
instead of plastic tube will ensure better heating of the gas
mixture.
[0088] The water surrounding the boxes had a constant and even
temperature. However, insufficient thermal insulation at the top,
and insufficient thermoconductivity at the bottom of the plastic
boxes resulted in 0.5-1.degree. C. differences between the
temperature of the water outside and the air inside the box (at
39.5.degree. C. water temperature). The magnitude of this
difference depended on the room temperature.
[0089] Temperature recovery to 0.5.degree. C. below the original
level needed 4-10 minutes after a short opening the door, and 12-30
minutes were required for recovery to the original level. Using
metal boxes recovery is expected to be much quicker (2-3 minutes
and 5-9 minutes, respectively).
[0090] In the Salvis Thermocenter, the temperature in the boxes was
appr. 0.5.degree. C. below the adjusted level. Recovery rates did
not differ from those observed using the plastic boxes and the
LBI.
[0091] Results of Embryo Culture Experiments
[0092] When used for embryo culture, the water temperature of the
LBI was adjusted to 39.5.degree. C. In this way the air temperature
of the boxes was between 38.5.degree. C. and 39.degree. C. Salvis
Thermocenter was adjusted to 39.degree. C. In this way the air
temperature of the boxes was about 38.5.degree. C. Results of Pilot
experiment 1 are shown in FIG. 9.
[0093] Cumulative results achieved in eight replicates were 145
blastocysts/333 oocytes (44%) and 255 blastocysts/567 oocytes (45%)
in the LBI and Salvis incubator, respectively.
[0094] Conclusion:
[0095] There was no significant difference between the quantitative
results achieved by the two thermostates. The blastocysts ratios
achieved were among the highest published so far (Brackett and
Zuelke, 1993; Farin et al., 1995; Hernandez-Lendezma et al., 1995).
When evaluating the quality of embryos by morphological criteria
using stereo microscopy, no differences between the two groups
could be revealed. In two cultures of one experiment (code 8),
hatching rate was followed until Day 10 and was 100% (22/22) and
91% (21/23) in the WBI and the Salvis group, respectively.
[0096] Pilot Experiment 2.
[0097] Bovine oocytes were collected from slaughterhouse-derived
oocytes, matured and fertilized in vitro as described in Pilot
experiment 1. After 22 to 32 h cocultivation of gametes
(fertilization) embryos were vortexed, then randomly distributed
into groups and cultured on the granulosa cell monolayer formed
spontaneously in the maturation four-well dishes (Nunc, Denmark) in
TCM-199 medium supplemented with 5% (10% from Day 4) calf serum.
Cultures were evaluated on Day 8. For maturation and fertilization,
K-system incubators were used (see Pilot experiment 1). Embryo
cultures were performed in 10.times.11 cm laminated foil sacks
(SFK, laminate foil 3370 UBA, 12 micron PETP Metal/75 micron PE
Gold) filled with expiration air, heat-sealed and submerged into a
38.7.degree. C. water bath. The depth of submerging on Day 1 to Day
4 (Day 0=day of insemination) was either 1 cm or 5 cm under the
water level in Pilot experiment 2a, and either 1 cm, 5 cm, 9 cm or
13 cm in Pilot experiment 2b. All bags were immersed to 1 cm under
the water level in both experiments on Day 4 to Day 8.
[0098] Results:
[0099] As shown in FIG. 10, significant (P<0.05 by Chi-square
test) increase of blastocyst/oocyte rates was achieved, when
embryos were subjected to 5 centimeter water pressure during the
first 4 days of development (Pilot experiment 2a). Cumulative rates
of 15 replicates were 329 blastocysts/365 oocytes (49%) and 262
blastocysts/599 oocytes (44%) in 5 and 1 centimeter water pressure,
respectively.
[0100] Further increase of the pressure did not improve
developmental rates. However, as shown in FIG. 11, increase of the
pressure up to 9 water cm did not impair developmental rates (Pilot
experiment 2b).
[0101] Conclusion:
[0102] The results of Pilot experiment 2 have proven, that the
slight increase of pressure in the foil bag submerged into water
had no detrimental effect on embryos up to the suggested maximum
depth (9 centimeter water). In contrast, a slight increase of
embryo developmental rates could be achieved by a moderate increase
of the pressure on Day 1-Day 4 of embryo culture.
[0103] Pilot Experiment 3.
[0104] Bovine oocytes were collected from slaughterhouse-derived
oocytes, matured and fertilized in vitro as described in Pilot
experiment 1. After 22 to 32 h cocultivation of gametes
(fertilization) embryos were vortexed, then randomly distributed
into groups and cultured on the granulosa cell monolayer formed
spontaneously in the maturation four-well dishes (Nunc, Denmark) in
TCM-199 medium supplemented with 5% (10% from Day 4) calf serum.
Cultures were evaluated on Day 8. For maturation and fertilization,
K-system incubators were used (see Pilot experiment 1). Embryo
cultures were performed in 10.times.11 cm laminated foil sacks as
described in Pilot experiment 2, filled either with a mixture of
3.5% or 5% carbon dioxide in air, heat-sealed and submerged into a
38.70.degree. C. water bath.
[0105] Results:
[0106] As shown in FIG. 12, significant (P<0.05 by Chi-square
test) increase of blastocyst/oocyte rates was achieved, when
embryos were cultured in 3.5% carbon dioxide. Cumulative rates of 9
replicates were 417 blastocysts/856 oocytes (51%) and 366
blastocysts/856 oocytes (43%) in 3.5% and 5% carbon dioxide in air,
respectively.
[0107] Conclusion:
[0108] The results of Pilot experiment 3 have proven, that slight
changes in composition of the atmospheric gas mixture may influence
the development of bovine pre-implantation embryos. In our culture
system, the 3.5% carbon dioxide in air mixture resulted in
significant increase in the developmental rates. The foil bag
system was a simple and useful tool in which these investigations
could be performed.
[0109] Pilot Experiment 4.
[0110] Bovine oocytes were collected from slaughterhouse-derived
oocytes, matured and fertilized in vitro as described in Pilot
experiment 1. After 22 to 32 h cocultivation of gametes
(fertilization) embryos were vortexed, and cultured on the
granulosa cell monolayer formed spontaneously in the maturation
four-well dishes (Nunc, Denmark) in TCM-199 medium supplemented
with 5% (10% from Day 4) calf serum. Cultures were evaluated on Day
8. For maturation and fertilization, K-system incubators were used
(see Pilot experiment 1). Embryo cultures were performed in
10.times.11 cm laminated foil sacks as described in Pilot
experiment 2, filled either with a mixture of 4% carbon dioxide in
air (Pilot experiment 4a) or with expiration air from one person
(Pilot experiment 4b), were heat-sealed and submerged into a
38.70.degree. C. water bath.
[0111] Results:
[0112] As shown in FIG. 13, no significant (P>O.l by Chi-square
test) difference of the developmental rates of the two groups in
Pilot experiment 4a was observed. Cumulative rates of 6 replicates
were 136 blastocysts/294 oocytes (46 %) and 134 blastocysts/285
oocytes (47%) in 4% carbon dioxide in air and expiration air,
respectively. In FIG. 14, results of 25 embryo culture experiments
(Pilot experiment 4b) are summarized. The cumulative developmental
rate was 517 blastocysts/1052 oocytes (49%).
[0113] Conclusion:
[0114] The results of Pilot experiment 4a and 4b have proven, that
expiration air is suitable to replace the industrial gas mixture,
and in a large series of experiments, high and even embryo
developmental rates can be achieved by the simplest way of
production of the atmospheric gas for embryo cultures. To use
expiration air for embryo culture, the foil bag system was uniquely
suitable, as the filling was easy and the required volume was
low.
[0115] Pilot Experiment 5.
[0116] Bovine oocytes were collected from slaughterhouse-derived
oocytes, matured and fertilized in vitro as described in Pilot
experiment 1. After 30 to 32 h cocultivation of gametes
(fertilization) embryos were vortexed, and cultured on the
granulosa cell monolayer formed spontaneously in the maturation
four-well dishes (Nunc, Denmark). Before use, the outer frame of
the dishes was cut, and the remaining 4.5.times.4.5.times.1 cm dish
was used for the experiments. Embryos were cultured in TCM-199
medium supplemented with 5% calf serum. Cultures were evaluated on
Day 7. For maturation and fertilization, K-system incubators were
used (see Pilot experiment 1). Embryo cultures were performed in
6.5.times.7 cm laminated foil sacks filled with expiration air,
heat sealed and submerged into a Minitube transportable incubator
(Minitube GMBH, 8311 Tiefenbach, Germany; Ref. no. 19180/0000)
sealed previously to become waterproof, then filled with water.
[0117] Results:
[0118] Blastocyst/oocyte rates of three identical replicates were
20/44 (45%), 16143 (37%), and 22/52 (42%), respectively. Cumulative
developmental rate of the three replicates was 58/139 (42%).
[0119] Conclusion:
[0120] The results of Pilot experiment 5 have proven, that the
foil-bag system combined with the use of expiration air is suitable
to establish suitable culture conditions at on field situations for
as highly sensitive objects as in vitro fertilized bovine embryos.
This culture can be maintained for 7 days and results in good
developmental rates. So far, no tissue culture equipment fulfilling
these requirements has been available.
[0121] Pilot Experiment 6.
[0122] Bovine oocytes were collected from slaughterhouse-derived
oocytes, matured and fertilized in vitro as described in Pilot
experiment 1. After 30 to 32 h cocultivation of gametes
(fertilization) embryos were vortexed, and cultured on the
granulosa cell monolayer formed spontaneously in the maturation
four-well dishes (Nunc, Denmark). Embryos were cultured in TCM-199
medium supplemented with 5% calf serum. Cultures were evaluated on
Day 8. For maturation and fertilization, K-system incubators were
used (see Pilot experiment 1). Embryo cultures were performed in
10.times.11 cm transparent laminated foil sacks (Rexam Metallising,
Camclear Polyester Laminate 12/45, Anti-Fog). The inner surface of
the foil was moistened by sterile paraffin oil, and the foil was
kept attached to the bottom of the dish, to increase optical
clarity. Sacs were filled with expiration air, then sealed. In
Pilot experiment 6a, foil sacs were immersed into a 38.6.degree. C.
water bath, but during the 7 days of the culture, 5 times taken out
of the water bath, placed on heated stage of an inverted
microscope, observed and photographed (each investigation was
performed in less than 5 min). In Pilot experiment 6b, the
transparent foil bag was wrapped again in laminated foil described
in Pilot experiment 2, then placed on the heated stage of an
inverted microscope, covered with a transparent plastic box
(16.times.16.times.4 cm, without bottom sheet) and cultured for
nine days.
[0123] Results:
[0124] Blastocyst/oocyte rates of four identical replicates in
Pilot experiment 6a were 20/39 (51%), 18/46 (39%), 18/41 (44%),
24/52 (46%), respectively. Cumulative developmental rate of the
four replicates was 45%. In Pilot experiment 6b, embryos cultured
in double sacks on the microscope stage reached the
blastocysts/hatched blastocysts stage without remarkable
morphological alterations.
[0125] Conclusion:
[0126] The transparent foil has been proven suitable for
maintaining the atmospheric conditions of the culture and for
visualization of the embryos under inverted microscope. In Pilot
experiment 6a, the repeated investigations did not result in severe
impairment in the development of bovine preimplantation embryos. In
Pilot experiment 6b, a continuous on stage microscopic
investigation of the development of embryos from one cell stage
till the hatched blastocyst stage was possible. Thus, the foil bag
system has been proven to be a simple, inexpensive and efficient
tool for repeated or continuous investigation of bovine embryo
development.
[0127] Conclusion
[0128] The suggested Liquid Bath Incubator is a basically new
solution for culturing sensitive cells and tissues. So far known,
this type of incubator is not included in the production list of
major incubator-producing companies (Heraeus, New Brunswick, Heto,
Jouan, Forma Scientific etc.) and no scientific publication deals
with this idea. LBI offers the following advantages:
[0129] 1. Temperature stability, quick recovery after opening, no
possibility of overheating.
[0130] 2. Gas mixture stability, quick recovery after opening.
[0131] 3. Humidity stability.
[0132] 4. Flexibility in using different gas mixtures
[0133] 5. Flexibility in using different atmospheric pressure
[0134] 6. Safety of operation: the only source of trouble is the
circulator, which is generally regarded as one of the most
trouble-free laboratory equipments.
[0135] 7. Cost-efficiency price
[0136] 8. Economy at work: very few gas mixture is needed,
maintenance costs and work are minimal
[0137] 9. Less danger of contamination, less problem with
cleaning.
[0138] 10. Transportability
[0139] 11. On-field establishment of tissue culture environment
[0140] 12. Possibility for frequent or continuous microscopic
observation.
[0141] In conclusion, the theoretical background and the
comparative characterization of the suggested incubation system can
be summarized in the following two tables:
1 Provided in vitro by Cells and tissues require during development
media incubator water, salts, nutrients, amino acids, + hormones,
growth factors defined pH (CO.sub.2/bicarbonate buffer system) + +
defined O.sub.2 + constant temperature + Commercially Suggested
Characteristics of an ideal IVF incubator available LBI constant
temperature + ++ constant gas phases (CO.sub.2, O.sub.2) + ++ high
speed of recovery of parameters + +++ realiability + +++
flexibility + +++ cost-efficiency (purchase, function) + +++ easy
operation ++ + easy cleaning sterility + ++ transportability - ++
on-field establishment of cultures - ++ microscopic follow-up +
+++
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